Andrea Rivero Arze

Nicolas Manier

Isabelle Metais

Catherine Mouneyrac

Amélie Chatel

Interaction of nanomaterials with freshwater organisms from 2 trophic levels II Posters

Keywords: Daphnids, nanomaterials, metallic oxides, transcriptomics

Participation as scientific communicator in the "Skype a scientist" initiative by Skype. Three interventions in 2020.

-Participation in the "European Researchers Night" with a radio interview about my research subject, that went on air November 17 th of 2020 at 8 pm, on Radio Alpa -Le Mans.

-Oral communications giving 2 workshops for the "MentorHer" initiative on the subject: "Scientific publications: how to read and write scientific papers

List of figures

 [START_REF] Sun | Comprehensive probabilistic modelling of environmental emissions of engineered nanomaterials[END_REF]. .. 15 [START_REF] Gottschalk | Environmental concentrations of engineered nanomaterials: Review of modeling and analytical studies[END_REF]. Sources of data 1. [START_REF] Mueller | Exposure Modeling of Engineered Nanoparticles in the Environment[END_REF]; 2. [START_REF] Gottschalk | Modeled Environmental Concentrations of Engineered Nanomaterials (TiO2, ZnO, Ag, CNT, Fullerenes) for Different Regions[END_REF]; 3.(O'Brien and Cummins, 2010); 4. [START_REF] Musee | Simulated environmental risk estimation of engineered nanomaterials: A case of cosmetics in Johannesburg City[END_REF]; 5. [START_REF] Gottschalk | Engineered nanomaterials in rivers -Exposure scenarios for Switzerland at high spatial and temporal resolution[END_REF]; 6.(A. C. Johnson et al., 2011); 7. [START_REF] Neal | Titanium in UK rural, agricultural and urban/industrial rivers: Geogenic and anthropogenic colloidal/sub-colloidal sources and the significance of within-river retention[END_REF]8.(A. Johnson et al., 2011). ........................... 17 Table 1: Values of 72h EC50, EC20 and IC50 of some metal oxide NMs on algae. Modified from [START_REF] Nguyen | Microalgal ecotoxicity of nanoparticles: An updated review[END_REF], [START_REF] Wang | TiO2 nanoparticles in the marine environment: Physical effects responsible for the toxicity on algae Phaeodactylum tricornutum[END_REF] and [START_REF] Saxena | Nanoecotoxicological Reports of Engineered Metal Oxide Nanoparticles on Algae[END_REF]

List of tables

Résumé de thèse

En raison de leurs propriétés particulières, l'utilisation de nanomatériaux manufacturés (NM) connait depuis plus de 15 ans un essor considérable, quel que soit le domaine industriel concerné. En parallèle, compte tenu des quantités grandissantes de nanoparticules ou nanomatériaux mises sur le marché, il convient d'anticiper une production de ces substances toujours plus variées notamment en ce qui concerne les paramètres physico-chimiques tels que la forme, la taille initiale de la particule ou encore la fonctionnalisation de celle-ci à l'aide de « coating » (enrobage). Ces propriétés uniques et spécifiques soulèvent cependant un certain nombre de questions, notamment en ce qui concerne leur devenir dans les compartiments environnementaux et leurs effets potentiels vis-à-vis des organismes aquatiques.

Au cours de ces dernières décennies, de nombreuses études d'écotoxicité ont été réalisées pour tenter d'analyser le comportement et les effets des NM sur les organismes d'eau douce. Cependant, il s'agit le plus souvent d'études au cas par cas et si ces études apportent de la connaissance sur les effets potentiels de ces substances, il existe encore aujourd'hui de nombreuses lacunes, en particulier en ce qui concerne les interactions NM-organismes, et les relations qui peuvent exister entre les caractéristiques physico-chimiques des NM et les caractéristiques biologiques des organismes dans la survenue d'effets toxiques. En effet, l'une des singularités de ces substances est leur dualité d'action, à la fois chimique et physique (i.e. adhésion, adsorption, altération physique des tissus). Dans la chaîne des événements qui conduisent à l'apparition d'un effet toxique, l'interaction entre un NM et un organisme biologique peut être perçue comme l'un des événements clés. Par conséquent, la compréhension des relations qui existent entre les propriétés des NM, les phénomènes d'interaction physique (adhésion, internalisation) et les effets potentiels chez les organismes apparaît primordiale dans un contexte de caractérisation a priori des dangers pour l'environnement et de réflexions pour la catégorisation de ces substances dans une finalité réglementaire.

La réglementation actuelle des NM considère que les similitudes moléculaires seules ne sont pas suffisantes pour justifier le regroupement et la catégorisation des NM. Ainsi, la nécessité de développer des approches qui incluent d'autres caractéristiques liées aux « particules » telles que la taille, la forme, la charge de surface, etc. et leurs modes d'action de toxicité, est soulignée par les principaux organismes de réglementation dans le monde. XIII Dans ce contexte, le travail réalisé dans le cadre de cette thèse a pour objectif d'étudier de façon systématique un ensemble de douze NM ayant des caractéristiques physico-chimiques variées et d'en analyser les interactions (adsorption, absorption, élimination et effets) sur différents organismes d'eau douce représentatifs des producteurs primaires (microalgues) et consommateur primaire (microinvertébrés). Les NM étudiés pour ce travail sont des oxydes métalliques provenant principalement du Centre Commun de Recherche de la Commission Européenne. Le set de NM est constitué de six dioxydes de titane (TiO2), un oxyde de zinc (ZnO), trois dioxydes de silicium (SiO2) et deux dioxydes de cérium (CeO2).

Dans une première partie, l'interaction des NM avec trois espèces différentes de microalgues (R. sucapitata, D. subspicatus et C. vulgaris) a été étudiée. Un test de contact à court terme a été développé pour cette finalité. L'interaction a été déterminée comme dépendante (i) des espèces d'algues et (ii) du type de NM. Les principales caractéristiques des NM qui ont joué un rôle dans l'interaction avec les microalgues sont la composition, la charge de surface et la phase cristalline. Dans une troisième partie, les effets de l'exposition de D. magna aux NM ont été étudiés par analyse transcriptomique. Les différences dans l'expression des gènes ont été analysées pour les processus cellulaires et individuels dans lesquels les NM ont des interactions connues. Ces processus sont : l'endocytose, le stress oxydatif, la détoxication des métaux, la réparation de l'ADN, le développement et la mue, la reproduction et la digestion. Une première série d'analyse en fin de phase d'exposition a permis de déterminer l'état de stress des organismes au point de rétention le plus élevée de NM. Une seconde série d'analyse à la fin de la phase d'élimination de 5 jours, a permis de déterminer les effets résiduels et la capacité de résilience de ces organismes après exposition aux NM. Ce travail a finalement permis de différencier les NM XIV par les effets de leurs mécanismes moléculaires sur les daphnies et d'établir des relations avec leurs caractéristiques physico-chimiques.

Enfin, dans une quatrième partie, la possibilité d'établir des relations entre les propriétés physico-chimiques des NM et leur comportement vis-à-vis des organismes aquatiques (attachement, absorption, l'élimination et toxicité) a été étudiée. Cette dernière partie a pour objectif de renseigner sur l'intérêt des paramètres biologiques étudiés et leur groupement afin de combler les lacunes existantes et de soutenir l'évaluation des risques des NM.

Introduction

Even if it is not clear when humans have started to take advantage of the properties of nanosized materials, it is certain that from the 60's the interest in studying and developing smaller and smaller objects began to gain momentum, starting with the famous lecture of the Nobel Prize winner Richard Feynman "There is plenty of room at the bottom" [START_REF] Feynman | There's Plenty of Room at the Bottom[END_REF]. However, it was not until the 90's that the emergence of appropriate methods allowed to develop what we now know as "nanotechnology" and that is considered as the technological revolution of the 21 st century [START_REF] Roco | International strategy for Nanotechnology Research[END_REF]. The prefix "nano" means a billionth (1*10 -9 ), and nanotechnology deals with matter having at least one dimension in the scale of a billionth of a meter (nanometer), which are called nanomaterials (NMs) [START_REF] Poole | Introduction to nanotechnology[END_REF].

Because of their nano-size, NMs have unique mechanical, catalytic, optical and electrical properties; that lead them to be employed in a wide range of applications including aviation and space, electronics, optics, textiles, medical devices, pharmaceuticals, cosmetics, food packaging, water treatment technology, building/construction industry, fuel cells, batteries, catalysts, biosensors and agents for environmental remediation (Klaine et al., 2008). Among all the diversity of existing NMs, metallic oxides NMs occupy the first position in terms of economic importance [START_REF] Luther | Industrial application of nanomaterials-changes and risks: Technology analysis[END_REF] and their physical, chemical and electrical characteristics, make them the most diverse class of materials covering almost all aspects of material science [START_REF] Fierro | Metal oxides: chemistry and applications[END_REF].

With the production of NMs on the rise, their release into the environment becomes inevitable and the NMs emissions may occur throughout their entire life cycle. Along with the release of NMs in the environment, the concerns of their impacts on the biota increases. The NMs inherent properties, such as particle size, aggregation/dispersion state, ions-release capacity, concentration, surface chemistry, and photo-chemical properties affect their performance, which may, therefore, influence their toxicity [START_REF] Nguyen | Microalgal ecotoxicity of nanoparticles: An updated review[END_REF]. In the aquatic compartment, the behavior of the NMs is intrinsically related to their interaction with aquatic organisms. That is why it is important to understand the relationship between the NMs characteristics and behavior in the environment with the NMs toxic effects for ecotoxicological studies and environmental risk assessment [START_REF] Corsi | Eco-Interactions of Engineered Nanomaterials in the Marine Environment: Towards an Eco-Design Framework[END_REF].

Under the Registration, Evaluation and Authorization of Chemicals (REACH) regulation, in the European Union it is necessary to register and to provide a detailed report of risks for every substance manufactured at 1 ton per year or more; and NMs are covered by the definition of a "substance" in REACH. (https://echa.europa.eu/regulations/nanomaterials).

However, many of the NMs on the market are not being produced at 1 ton/year and, as such, do not require intensive analysis of risk. The lack of NMs analysis drives to a lack of NMs knowledge, which makes the development of a NMs regulatory policy even more difficult [START_REF] Barrick | Ecotoxicological study of the impact of nanomaterials on marine mussels through a high throughput screening approach[END_REF].

To produce the report of risk, it is necessary to test the NMs, but case-by-case study of all the different existing and new NMs would require a lot of time and resources. Thus, grouping and read-across strategies are needed for NMs risk assessment. These approaches are already established for chemical substances (ECHA, 2008) (OECD, 2007); but for NMs further aspects need to be considered, since approaches based on molecular structural similarity alone are not sufficient to develop and verify grouping/read-across hypothesis that allow an adequate hazard assessment of NMs (Schwirn and Völker, 2019). That is why, the European Chemical Agency (ECHA) has developed nano-specific guidance for grouping considering properties beyond chemical composition (particle size, shape, or solubility), and highlights the importance of toxicokinetic studies in grouping, read-across, and for in vitro to in vivo extrapolation (ECHA/JRC/RIVM, 2016). New concepts are also being developed to improve the integration of mechanistic information, such as the adverse outcome pathways (AOP) concept, or even high-throughput screening (HTS) and omics data (Mech et al., 2019).

One of the singularities of the NMs is their duality of action, both physical (i.e. adhesion, adsorption, physical alteration of tissues) and chemical (ion release, reactive oxygen species generation). In the chain of events leading to the occurrence of a deleterious effect, the interaction between a NM and a biological organism can be seen as one of the key events.

Consequently, the understanding of the links between the NMs properties, the phenomena of physical interaction (adhesion, internalization) and the potential effects in organisms seems essential in a context of a priori characterization of the NMs environmental hazards for the NMs categorization with regulatory purposes.

In this context, the main objective of this thesis is : To accomplish this goal, a set of metallic oxide NMs with different physicochemical characteristics was chosen to be studied systematically. Then, employing this set of NMs, the following specific objectives are established:

To investigate the characteristics of the interactions between

• To develop a method that allows the qualitative and quantitative determination and comparison of the interaction of NMs and three different species of freshwater microalgae, and to analyze the factors that influence those interactions.

• To determine the NMs uptake and elimination behaviors in the microcrustacean D. magna by stablishing the kinetic parameters that rule those behaviors and their links with the NMs properties.

• To analyze the NMs early molecular effects at the scale of the genetic expressions of D. magna organisms during the exposition to NMs and after an elimination period.

• To analyze the relevance of the parameters involved in the nano-bio interactions in the clustering and potential grouping of NMs, by studying the uptake and elimination kinetics, the early molecular effects along with the pristine and media dependent NMs characteristics.

The different parts of this thesis manuscript have been conceived to respond to the proposed objectives, in that context the present document is constitute by the following chapters:

This manuscript will begin with a bibliographic study. Through Chapter 1, principal concepts involved in the NMs characteristics, production, applications, emission, socioeconomic importance and regulation problematics are developed. Furthermore, descriptions of their known interactions with biological systems and toxic effects are presented.

In Chapter 2, consisting of the materials and methods part, the biological models and the NMs protagonists of this research work are described. The experimental designs, as well as the employed and developed methods are fully detailed.

The first axis of this research constitutes Chapter 3, in which the interaction between the primary producers, three freshwater microalgae species, with the set of selected NMs is studied through a "short contact test" method specially developed for this purpose.

The NMs interaction with the next trophic level, the primary consumers, represented by the microcrustacean Daphnia magna is addressed in Chapter 4. NMs uptake and elimination behaviors and kinetics are determined. Additionally, the early molecular effects in these organisms during the exposure to NMs and the resilience or effects after the NMs elimination are investigated in Chapter 5 through transcriptomic analyses.

Wrapping all the precedent information, Chapter 6 analyses the influence of the NMs physicochemical characteristics, the nano-bio interactions with algae and the uptake and elimination kinetics and differential genetic expressions in daphnids; to distinguish groups of NMs that can be described by similar interrelationships of these variables.

A final Chapter will present the general conclusions of this work, along with the perspectives for further and complementary research.

Chapter 1: Scientific context 1. The "nano world"

Historically, humans have sought to understand the world they live in; in doing so, they have studied the smaller and smaller constituents of life, and the aim is to always go beyond what is already known. Every time the knowledge goes deeper, it reveals that there is more and more to discover. As the Nobel Prize winner in physics, Richard Feynman, had said "There is plenty of room at the bottom" [START_REF] Feynman | There's Plenty of Room at the Bottom[END_REF].

The prefix "nano", derived from the Greek "nanos", signifies "dwarf". Nano is now a common label for many concepts associated to modern science, where many nano-related words have recently appeared in dictionaries, including nanometer, nanoscale, nanoscience, nanotechnology, nanostructure, nanotube, nanowire, and nanorobot [START_REF] Buzea | Nanomaterials and nanoparticles: Sources and toxicity[END_REF].

The nanometer is a metric unit of length and denotes one-billionth of a meter or 1x10 -9 m. A lot of things occur on this scale in nature: it is the scale where groups of atoms come together to form molecules and proteins and it is the scale of their interactions. On this scale we know that matter can be manipulated, atom by atom, to construct it and to improve its chemical, physical or electronic properties (CEA, 2010).

The world of the infinitely small is governed by completely different laws from the one at the macroscopic scale. Effects negligible at the macroscopic level become important at the nanometer scale, and vice versa. Additionally, quantum effects can begin to dominate the behavior of matter at the nanoscale (particularly at the lower end) affecting the optical, electrical and magnetic behavior of materials [START_REF] Wautelet | Introduction to the Nanoworld[END_REF].

Nanotechnology

Nanotechnology, as defined by the United States National Nanotechnology Initiative, is science, engineering, and technology conducted at the atomic, molecular, or macromolecular scale, leading to the controlled creation and use of structures, devices, and systems with a length scale of 1 to100 nanometers [START_REF] Nnco | National Nanotechnology Initiative Strategic Plan[END_REF]. It exploits the unique chemical, physical, electrical, and mechanical properties that emerge when matter is structured at this size. Nanotechnology has an interdisciplinary nature; it can be found in the fields of physics, chemistry, environmental sciences, biology, medicine, as well as electrical and mechanical engineering [START_REF] Poole | Introduction to nanotechnology[END_REF].

The development of nanotechnology is considered the industrial revolution of the 21 st century [START_REF] Roco | International strategy for Nanotechnology Research[END_REF]. But nanotechnology raises many of the same issues as any new technology, including the concern about toxicity and environmental impact of nanomaterials.

Nanomaterials

Nanomaterials have existed in nature long before humans were aware of them.

Organisms have evolved in their presence and they have developed key mechanisms to accommodate to their occurrence. As such, nanoscale products are not inherently dangerous for organisms (Klaine et al., 2008). These nanometric objects are generated by natural phenomena such as volcanic eruptions or forest fires which produces nanoparticle dust and carbon black or mechanical wear which creates silica nanomaterials [START_REF] Griffin | Natural Nanoparticles: A Particular Matter Inspired by Nature[END_REF].

Humans have also involuntarily produced artificial nano-objects, long before talking about nanotechnologies. Indeed, at the time, craftsmen were already producing materials with remarkable properties by incorporating nanometric compounds. For example: certain stainedglass windows of the Middle Ages were made based on red glass containing nano precipitates of copper; or some Roman cups with specific optical properties contained metallic nanoparticles of gold and silver [START_REF] Mazten | MOOC Nano : Comprendre les nanosciences[END_REF].

Engineered or manufactured nanomaterials, that for the purpose of the present document, will be called from here on just nanomaterials (NMs); are defined under the very broad definition of any material that is <100 nm in size in at least one dimension [START_REF] Boverhof | Comparative assessment of nanomaterial definitions and safety evaluation considerations[END_REF]. The European Commission recommendation on the definition of nanomaterials (EC, 2011) defines them as:

"´Nanomaterial´ means a natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate or as an agglomerate and where, for 50% or more of the particles in the number size distribution, one or more external dimensions is in the size range 1 nm -100 nm. In specific cases and where warranted by concern for environment, health, safety or competitiveness the number size distribution threshold of 50% may be replaced by a threshold between 1 and 50%" Internationally, there is a wide array of different versions for the definitions of NMs, with many of them centered around production of objects within the 1-100nm size range. Some government bodies also use additional information including: how much of the material falls into the size category to be considered an NM, if the materials were intentionally produced, if they display novel properties when compared to the bulk material, if aggregations or agglomerations are considered and what it the solubility profile of the materials [START_REF] Barrick | Ecotoxicological study of the impact of nanomaterials on marine mussels through a high throughput screening approach[END_REF].

There are several important applications of nanomaterials in fields such as aviation and space, chemical industry, optics, fuel cell, batteries, sensors, power generation, aeronautic industry, building/construction industry, automotive engineering, consumer electronics, thermoelectric devices, pharmaceuticals, and cosmetic industry [START_REF] Ali | Application of Nanomaterials in Environmental Improvement[END_REF].

Production

As mentioned above, nanotechnology is applied to very diverse fields. Thus, specific techniques and specific instrumentation for fabrication, control and measurement have been developed. The main challenge during the fabrication of NMs is to control size, shape, structure and distribution. Concerning production methods, two main routes can be distinguished as described by [START_REF] Luther | Industrial application of nanomaterials-changes and risks: Technology analysis[END_REF]):

• Top-down approach: Reduction in structure sizes of bulk elements to the nanometer scale by applying specific machining and etching techniques. The biggest problem with this approach is the imperfection of surface structure. The main techniques are:

-Lithography: A variety of lithographic methods exists, which are based on different physical principles and have different characteristics with regard to resolution, velocity, patterning and transfer steps, such as beam lithography techniques (optical, x-ray-, ion beam or electron) or soft lithography techniques (printing, stamping, molding and embossing).

-Mechanical Milling: Mixtures of elemental or previously alloyed powders are ground using high energy impact forces and continuously refined until the nanometric size is attained.

-Electro-Explosion: Using a very strong current over a very short time in thin metal wires to create a plasma state which causes the resistivity of the wires to increase, resulting in a supersonic explosion -Laser Ablation: Use of a laser to boil off nanomaterials from the surface of a solid target. A broad range of nanoparticulate materials can be obtained by this method.

• Bottom-up approach: Controlled assembly of atomic and molecular aggregates into larger systems (e.g., clusters, organic lattices, supramolecular structures and synthesized macromolecules). The bottom-up approach generally produces nano structures with fewer defects as compared to the nanostructures produced by the top-down approach.

The main techniques are:

-Sol-gel: This process is well adapted to produce metal oxides which are made when a solution transitions to a gel. The process results in the precipitation of NMs.

-Aerosol: Consists on spraying solid or liquid precursor chemicals into a gas phase that is followed by a heating process (pyrolysis) and the formation of NMs.

-Chemical Vapor Deposition: Activation of a chemical reaction between a substrate and a gaseous precursor to produce the NMs. Process can be achieved through intense heat or through plasma. This technique is widely used to produce carbon nanotubes.

-Atomic or Molecular Condensation: Mainly used to produce metal containing NMs, where the bulk material is vaporized and then rapidly cooled, causing condensation and the formation of the NMs.

-Supercritical Fluid Synthesis: For this technique, a fluid is forced into a super critical state by regulating its temperature and its pressure, and rapid expansion results in the formation of the NMs.

-Electro Spinning: Diluted polymer solutions are charged with high voltage and fine jets of polymers release from the surface, forming a continuous web of fine fibers.

-Templates: Use of nano-sized pores in a surface as molds to form NMs. Artificial templates can be created on a plane surface or a gas-liquid interface by forming self-assembled monolayers.

-Self-Assembly: This technique is based on the selective attachment of certain types of molecules that will spontaneously align themselves to a specific surface including biomolecular recognition. The coated material can then in turn be the substrate for a layer of a different compound.

General classification

Due to the huge amount of NMs and the wide diversity of their characteristics, there are many classification systems applicable. The main categorizations commonly considered divide NMs by their dimensionality, composition, structure and way of production. Summarizing the most relevant classifications, NMs can be categorized as follows:

• On the basis of dimension: NMs can be classified as zero, one, two, and three dimensions as presented by [START_REF] Singh | Recent Advances on Classification, Properties, Synthesis, and Characterization of Nanomaterials[END_REF].

-Zero dimensions: These point-like particles (small as a point) are the most common type of NMs with all the dimensions within the nanoscale. Typical examples are quantum dots (QDs), hollow spheres and nano lenses.

-One dimension: These NMs have at least one dimension larger than nanoscales and the other dimensions are in the nano range; as nanofibers, nanotubes, and nanorods.

-Two dimensions: These NMs with plate-like structures are two dimensions larger than the nanoscale; as nanofilms, nanolayers, and nanocoating.

-Three dimensions: These NMs have all three dimensions larger than 100 nm but their components are below 100 nm in size. They are generally nonporous in nature and have many applications. Common examples are nanocomposites, bundles of nanofibers and multinanolayer-type structures.

• On the basis of composition: NMs can be made from carbon, inorganic, organic or composite compounds.

-Carbon-based: NMs made of carbon content. These NMs are subdivided in fullerenes, graphene, carbon nanotubes, carbon nanofibers and carbon black.

-Inorganic-based: NMs made of metals or metal oxides. Semiconductor NMs are also synthesized from silicon and ceramic materials.

-Organic-based: These NMs are made up of organic matter other than carbon. The noncovalent (weak) interaction applies in these types of materials.

-Composite-based: NMs made of more than one layer of nanoparticles. The composites may be made up of many types of materials such as metal, ceramic, organic, inorganic, carbon-based, or bulk polymers.

• On the basis of structure: This classification is detailed below, following what is presented by [START_REF] Luther | Industrial application of nanomaterials-changes and risks: Technology analysis[END_REF]):

• Nanoparticles: Constituted by tens or hundreds of atoms or molecules and can have a variety of sizes and morphologies (amorphous, crystalline, spherical, needles).

They are produced as dry powders or liquid dispersions.

• Nanowires and tubes: Linear nanostructures that are made from metals, semiconductors or carbon (single, multi-walled, filled or surface modified).

• Nanolayers: Surfaces and layers that can optimize the interface of different material classes when added to products • Nanopores: These NMs have defined pore-sizes in the nanometer range. The potential applications of these NMs include catalysis, thermal insulation, electrode materials, environmental filters and controlled release drug carriers.

• Drug carriers: A subclassification of emerging nanoforms specially employed in the drug delivery is summarized below as presented by [START_REF] Wakaskar | General overview of lipid-polymer hybrid nanoparticles, dendrimers, micelles, liposomes, spongosomes and cubosomes[END_REF]:

• Lipid-polymer hybrid nanoparticles: Consists on a polymer and a lipid component and possesses a core-shell structure. They are used to deliver proteins, hydrophilic and hydrophobic drugs.

• Dendrimers: These hyper-branched and predominantly 3D macromolecules, have a central core which consist on repeating units and many terminal functional groups that are located in the outer region of the macromolecular architecture.

• Polymeric micelles: These amphiphilic block copolymers are highly biostable and are particularly used to deliver extremely hydrophobic drugs (anti-cancer agents), contrast imaging molecules and peptides.

• Liposomes: These nanoparticulate structures made of biocompatible phospholipids are self-assemble into vesicles. They are used for active and passive drug delivery.

• Spongosomes and cubosomes: These membrane-type nanoparticles encapsulate hydrophobic chemotherapeutic agents. Cubosomes have a much more organized membrane structure than spongosomes.

All the previously presented NMs classification categories are summarized on Figure 1. The fast rate at which new NMs are being developed makes very difficult to maintain a stable categorization scheme. Thus, it is common to find materials that do not fall within the described categories.

Metal oxide nanomaterials

Metal oxide NMs occupy the first position in terms of economic importance within the range of inorganic nanomaterials [START_REF] Luther | Industrial application of nanomaterials-changes and risks: Technology analysis[END_REF]. The unique physical, chemical and electrical characteristics of these NMs make them the most diverse class of materials covering almost all aspects of material science in the fields of semi conductivity, superconductivity, ferroelectricity, and magnetism [START_REF] Fierro | Metal oxides: chemistry and applications[END_REF].

In nature, titanium dioxide (TiO2) exists in three different crystalline structures: rutile, anatase, and brookite. The anatase and rutile forms have natural and industrial importance, while the brookite is rarely used [START_REF] Shah | Hazardous Effects of Titanium Dioxide Nanoparticles in Ecosystem[END_REF]. Rutile TiO2 is a thermodynamically stable structure and it is used as a white pigment, a coating material for optical lenses and a dielectric material because of its high refractive index compared to other materials [START_REF] Lee | Synthesis of brookite TiO2 nanoparticles by ambient condition sol process[END_REF].

When the size of TiO2 NMs reduces, the anatase form becomes more stable providing greater number of oxygen vacancies on the surface than the rutile structure; which results in greater chemical and photocatalytic activities compared to rutile [START_REF] Dontsova | Metaloxide Nanomaterials and Nanocomposites of Ecological Purpose[END_REF]. Anatase TiO2 NMs are the mostly studied and synthesized nanostructures with wide applications in biological systems for cancer treatment, in photocatalytic reactions, sun blocking creams and in industry due to their versatility, selectivity, purity and homogeneity [START_REF] Shah | Hazardous Effects of Titanium Dioxide Nanoparticles in Ecosystem[END_REF].

Zinc oxide (ZnO) NMs have physical properties known as "quantum size effects".

Quantum confinement increases the energy gap of quasi-one-dimensional ZnO which is confirmed by photoluminescence. ZnO NMs have the richest family of nanostructures among all materials, which differ in structure and properties; and that are in demand in a wide variety of applications, such as in optoelectronics, sensors, transducers, and photocatalytic and biomedical materials [START_REF] Dontsova | Metaloxide Nanomaterials and Nanocomposites of Ecological Purpose[END_REF]. Amorphous silicon (SiO2) NMs are characterized to be very stable NMs and they have their applications as additives to cement, paint, cosmetics, food, and drug delivery devices; they are also used as additive in animal feed or as support for heterogeneous catalysts [START_REF] Maser | In vitro and in vivo genotoxicity investigations of differently sized amorphous SiO2 nanomaterials[END_REF]. It has likewise proved to be a very effective additive to polymers improving strength, flexibility, and durability [START_REF] Sobolev | Nanomaterials and Nanotechnology for Highperformance cement composites[END_REF]. Lately, NMs of silica with encapsulated DNA are been proposed for consumer goods tagging and for labelling of living organisms, which provides a valuable tool to ecological research when elucidating food web architecture or evaluating species habitat for conservation or sustainable use [START_REF] Stark | Industrial applications of nanoparticles[END_REF].

Cerium dioxide nanomaterials (CeO2 NMs) have their main application is in the field of catalysis for their ability to quickly and conveniently mutate of the oxidation state between Ce 4+ and Ce 3+ . They have also various engineering applications such as solid-oxide fuel cells, hightemperature oxidation protection materials and solar cells. Additionally, CeO2 NMs were recently found to have multi-enzyme mimetic properties that produce various biological effects. These NMs have emerged as a fascinating and lucrative material in biological fields such as bioanalysis, biomedicine, drug delivery, and bioscaffolding [START_REF] Xu | Cerium oxide nanoparticle: a remarkably versatile rare earth nanomaterial for biological applications[END_REF].

New uses of metal oxide NMs, especially TiO2, ZnO, SnO2, ZrO2, and Fe3O4 are becoming increasingly popular in applied ecology and electrochemistry. The main applications are sorbents, sensors (for volatile organic compounds or aqueous analytes), energy conversion (nanostructured electrocatalyst for photovoltaics and fuel cells), energy storage systems (nanostructured anode and cathode materials), electrochemical and photo-electrochemical water electrolyzer [START_REF] Maduraiveeran | Earth-abundant transition metal and metal oxide nanomaterials: Synthesis and electrochemical applications[END_REF] [START_REF] Dontsova | Metaloxide Nanomaterials and Nanocomposites of Ecological Purpose[END_REF].

Nanometrology

The necessity of an accurate and complete characterization and metrology of NMs is primordial in ecotoxicology testing, since it has been proven that the reactivity and toxicity of NMs depends on their physical and chemical properties. Depending on exposure conditions, NMs can exhibit different behaviors of aggregation, attachment to surfaces, release of toxic metal ions, complexation and interaction with environmental constituents as humic substances, polysaccharides and cellular debris. These conditions can affect their bioavailability, uptake and toxicity; defining the dose-response relationship between the NMs and the organisms [START_REF] Lead | Nanomaterials in the environment: Behavior, fate, bioavailability, and effects-An updated review[END_REF].

The most common methods to characterize NMs are listed below along with their advantages and limitations as described by (Hodoroaba et al., 2019):

• Dynamic light scattering (DLS) is frequently used for size measurement, and it works very well for simple samples that are monodispersed, but size data are often inaccurate in aggregating/aggregated systems.

• Scanning electron microscopy (SEM) is excellent for NMs size and shape characterization, because the sample preparation and image acquisition are relatively quick and simple. Although SEM images are 2D, it is possible to obtain a 3D representation through model-based measurements.

• Transmission electron microscopy (TEM) is a versatile technique to analyze the size, morphology, crystallography, structure and chemical composition of a wide range of NMs. It can provide a spatial resolution reliably covering the complete nanometric size range (1-100nm) and it can assess the agglomeration/aggregation state of NMs. The main limitations are presence of artifacts resulting from the vacuum conditions and the fact that the reliability of the analysis depends on the sample preservation through the preparation process. It remains very cost and labor intensive.

• Atomic force microscopy (AFM) is well suited for nanomechanical measures. For size measures, it is adequate to measure the height of a NMs deposited on a clean flat surface but there are uncertainties and artifacts in the lateral dimensions.

• Single particle inductively coupled plasma mass spectrometry (SP-ICP-MS) has proven to be a powerful technique that allows the single and joint analysis of particle number and concentration on an ion-specific basis. It requires minimal to no sample preparation.

The limitation of this method is the smallest size that can be quantified (20-40nm) and it is element dependent.

• Nano-Particle tracking analysis (NTA or PTA) is a common technique for rapidly and inexpensively characterizing size, concentration and zeta potential of NMs in a liquid suspension. The obtained size measures have a significantly higher resolution than DLS and it can discriminate the fluorescent NMs from a mix.

Zeta potential measurements can be achieved with a DLS or a PTA equipment by applying an electric field across the sample, using the technique of micro-electrophoresis. Zeta potential identifies the apparent surface charge of NMs and it is the key parameter that controls electrostatic interactions in particle dispersions [START_REF] Kaszuba | High-concentration zeta potential measurements using light-scattering techniques[END_REF].

It can be noticed that all methods have advantages and limitations, and that no single metrological method will give all the data required for a complete characterization of NMs.

Emission

With the production of NMs on the rise, their release into the environment becomes inevitable. Release of NMs into the environment may occur throughout their entire life cycle: from their production to the fabrication of NMs-containing products, to the use and end of life phase of those products (see Figure 2). Release of NMs can occur during their production and incorporation into products, but it mainly takes place during the use and disposal phases. The receptors of the NMs emission to the environment are the air, soil, water and sediments; and transport within environmental compartments occurs. Currently, there are almost no specific trace analytical methods available to quantify NMs in environmental samples. Therefore, to obtain information on existing levels of NMs in the environment and to perform a proactive environmental risk analysis, the only way is to model predicted environmental concentrations. To this end, information on the flows of NMs into the environment is particularly needed [START_REF] Sun | Comprehensive probabilistic modelling of environmental emissions of engineered nanomaterials[END_REF].

The [START_REF] Keller | Global life cycle releases of engineered nanomaterials[END_REF] and (Keller and Lazareva, 2014) studies provide the first global assessment of NMs emissions to the environment based on the life-cycle approach. It covers the top ten most produced NMs by mass, analyzed through their dozen major applications. The analysis combines NMs market information and material flow modelling. The estimated global mass flow of NMs is presented in Figure 3. The studies estimated that, for 2010, the NMs global release of NMs went up to 260,000-309,000 metric tons, from which 63-91% ended up in landfills, 8-28% in soils, 0.4-7% in water bodies and 0.1-1.5% in the atmosphere. (Keller and Lazareva, 2014).

Concerning the emissions to the surface waters, a study by [START_REF] Gottschalk | Environmental concentrations of engineered nanomaterials: Review of modeling and analytical studies[END_REF] modeled the concentrations that can be found in these ecosystems for the most common used NMs as presented in Figure 4. This model presents the predicted environmental concentrations of nano-TiO2 in rivers as a range from 3 ng/L to 1.6 mg/L; for nano-ZnO from 1 to 13 ng/L and for nano-CeO2 around 24.3 ng/L.

Environmental behavior and fate

The environmental fate and behavior of NMs is a fundamental factor to understand their effects on environmental health. It can allow to identify specific hazards and is a key aspect of environmental risk assessment. Knowledge of the sources, pathways, transformations and sinks for NMs provides information on the compartments (air, soil, water, sediment, and biota) that are exposed to the NMs [START_REF] Lead | Nanomaterials in the environment: Behavior, fate, bioavailability, and effects-An updated review[END_REF]. The distribution of NMs in the environmental compartments plus the main processes that occur on them, are summarized in Figure 5.

Transformations of NMs in the environment are analogous to the problem of changes in speciation in metal fate, behavior, bioavailability and effects. These transformations can be subdivided into physical (aggregation, agglomeration, sedimentation, and deposition), chemical (dissolution and subsequent speciation changes, redox and photochemical reactions, and corona formation), and biological processes (biodegradation and biomodification) [START_REF] Lead | Nanomaterials in the environment: Behavior, fate, bioavailability, and effects-An updated review[END_REF]. Aquatic ecosystems are the main recipient of all contaminants, including NMs. The most important process of NMs in these ecosystems, as described by [START_REF] Zhang | The effects and the potential mechanism of environmental transformation of metal nanoparticles on their toxicity in organisms[END_REF], are: i) NMs aggregation and/or agglomeration which increases the size of NMs and decreases their concentration in solution, accelerating their deposition. This process is facilitated by the great number of macromolecules in the aquatic systems that would most likely interact with the NMs.

ii) NMs dissolution, that has a profound effect on their transport, distribution and bioavailability. iii) Redox reactions, which depend on the surface chemistry of the NMs and on the oxygen level in the environment. Redox can, in turn, affect dissolution, stability and association behaviors. iv) Biological transformations that are mainly sulphidation and bacterial oxidation and degradation of surface coatings.

Another NMs transformation process in the environment is the formation of eco-coronas that are formed by extracellular proteins and polymeric substances, also by tanic and humic acids; that can increase the NMs suspension stability (Lynch et al., 2014). Additionally, photocatalytic activity of NMs in aquatic systems can generate reactive oxygen species (ROS) [START_REF] Schultz | Aquatic toxicity of manufactured nanomaterials: challenges and recommendations for future toxicity testing[END_REF].

Environmentally transformed NMs can exhibit totally different toxicity than the original NMs (see Figure 6).

Socio-economic aspects

Nanotechnology is a universal technology with enormous breadth which can be applied to virtually any manufactured artefact [START_REF] Ramsden | Applied Nanotechnology: The Conversion of Research Results to Products[END_REF]. This potential, that was recognized several years ago by governmental institutions, promoted the deployment of strategies oriented to the creation of the basis for nanotechnology further industrial exploitation in order to provide a large socioeconomic impact [START_REF] Matteucci | Deployment and exploitation of nanotechnology nanomaterials and nanomedicine[END_REF].

According to a report issued by (Zion Market Research, 2020), the global NMs market accounted for USD 13.6 Billion in 2020 and are expected to reach USD 26.3 Billion by 2028, growing at a compound annual growth rate of around 8.1% between 2021 and 2028. Growing governmental and public funding of NMs exploration and production operations is anticipated to favor up the worldwide trends over the approaching years. The escalating usage of the merchandise in healthcare devices, transport, packaging, construction activities, and electrical and electronics appliances will stimulate this expansion.

The report identifies: BASF, Evonik Industries, Covestro, Altair Nanotechnologies Inc., EMFUTUR Technologies, and SkySpring Nanomaterials as some of the major players in the global NMs market. Concerning to regions, North America dominates the market mainly due to the increasing demand from the packaging industry and to the extensive application of nanotubes in various industries. Europe is also a leading actor accounting for a significant market share while an increasing demand is expected in the upcoming years due to several emerging applications in the European healthcare industry.

Nanotechnology has substantially improved the way we live. The technological innovations based on NMs boosted the production of more potent and more accessible electronics. The applications in food production allow to produce high quality foods in a more sustainable way. In the medical field it has been used in diagnosis and treatment of cancer, helping to save lives and improving living conditions for patients. Environmental benefits come with the development of water depollution techniques and energy production and storage.

But advances in nanotechnology also present numerous challenges and risks in health and environmental areas. The biggest question is how to deal with uncertainty and risk assessment in this developing discipline [START_REF] Khan | Ethical and social implications of nanotechnology[END_REF]. And if nanotechnology becomes as fruitful as some expect, harmful outcomes are inevitable. The social implications of these outcomes encompass many fundamental areas such as ethics, privacy, environment, and security. The issues to handle would include how to safeguard privacy in a world with nanosnooping devices, to what extent the manipulation of human beings should be permitted, and how to minimize the risk of runaway nanobots [START_REF] Moor | Nanoethics: assessing the nanoscale from an ethical point of view[END_REF].

Faced with scientific uncertainties and societal questions raised by NMs, the French Agency for Food, Environmental and Occupational Health & Safety (ANSES) created a dialogue committee on "Nanomaterials & health" in 2012. This dialogue committee is a forum for discussions with stakeholders on current scientific work and research needs. It also enables the Agency to be informed of civil society's expectations and concerns in this area and helps guide its work programs (ANSES, 2020).

To date, the European Union Observatory for Nanomaterials (EUON) database contains 340 registered substances (https://euon.echa.europa.eu/). In France, producers, importers and dealers must declare the products containing NMs in the "R Nano" database, held by the ANSES agency. Currently the most comprehensive inventory of products containing NMs available is "The Nanodatabase" (https://nanodb.dk/en/), developed by the DTU Environment, the Danish Ecological Council and the Danish Consumer Council. However, most of the provided NMs, have little to no information associated with them. To October 2021, this database references 5207 products containing NMs, and 70.4% of them have unknown compositions, which poses a problem as it impedes the ability to develop a comprehensive framework for regulation.

Regulation of nanomaterials and grouping

Following the Precautionary Principle, that provides a fundamental policy basis to anticipate, avoid and mitigate threats to the environment; policies concerning NMs have been intended by regulatory bodies throughout the world [START_REF] Iucn | Guidelines for applying the Precautionary Principle[END_REF]. Still, the European Chemical Agency (ECHA) and the Environment Protection Agency (EPA) Toxic Substance Control Act (TSCA) currently do not have a specific regulatory policy to address nanomaterials [START_REF] Breggin | Securing the promise of nanotechnologies: towards transatlantic regulatory cooperation[END_REF].

The Organization for Economic Co-operation and Development (OECD) conducted a study in 2006 to harmonize the testing and assessment of NMs between member nations (OECD, 2018). The study concluded that many currently existing guidelines for chemicals safety testing are suitable for NMs, but they may need to be adapted for some NMs specific properties. It also pointed out the difficulty to have a harmonized definition for NMs and this has impeded the generation of comprehensive regulatory policies.

In the European Union (EU), the Registration, Evaluation and Authorization of Chemicals (REACH) is the over-arching legislation applicable, to the manufacture, placing on the market and use of substances on their own, in preparations or in products. NMs are covered by the definition of a "substance" in REACH, even though there is no explicit reference to nanomaterials (ECHA, n.d.).

The REACH legislation obligates to register substances manufactured at 1 ton per year or more, and NMs enter under this obligation. This registration includes a detailed report of potential risks (ECHA, n.d.). Currently many of the NMs on the market are not being produced at 1 ton/year and, as such, do not require intensive analysis of risk. The lack of NMs analysis drives to a lack of NMs knowledge, which makes the development of a NMs regulatory policy even more difficult [START_REF] Barrick | Ecotoxicological study of the impact of nanomaterials on marine mussels through a high throughput screening approach[END_REF].

There are some specific guidelines concerning NMs under REACH, but this is currently being investigated in order to improve its efficacy [START_REF] Pettitt | Minimum physicochemical characterisation requirements for nanomaterial regulation[END_REF]. For example, one of the additional parameters proposed to be studied while performing ecotoxicological hazard assessment of chemicals, is to consider the physical interactions and effects of NMs; as indicated in the Appendix R7-1 for nanomaterials applicable to Chapter R7a (Endpoint specific guidance) in the Guidance on information requirements and chemical safety assessment published by the ECHA [START_REF] Echa | Topical Scientific Workshop on New Approach Methodologies in Regulatory Science[END_REF].

In order to improve the collective work on the NMs regulation issues, the European Union created the Nanosafety Cluster. This high-profile platform maximizes the synergies between European-level projects addressing the safety of NMs and technologies enabled by the use of nanoforms. The studied aspects, in line with the Horizon 2020 program, include toxicology, ecotoxicology, exposure assessment, mechanisms of interaction, risk assessment and standardization (EU, 2021).

• Grouping

A large number of NMs may share the same chemical composition but differ in particle size, shape, and surface chemistry; which can result in different exposure, toxicokinetic and hazard profiles (Mech et al., 2019). But testing every NM would demand substantial resources.

Therefore, regulators are searching to implement Intelligent Testing Strategies (ITS) to quickly screen a large amount of NMs in a cost-effective manner, and this can be achieved by applying grouping and read-across approaches [START_REF] Stone | ITS-NANO -Prioritising nanosafety research to develop a stakeholder driven intelligent testing strategy[END_REF].

For chemicals in general, grouping and read-across are well-established regulatory concepts to minimize the need for specific testing. They are based on hypotheses that describe how specific hazards can be predicted based on a set of known physicochemical properties and toxicity testing (Stone et al., 2020). However, the amended REACH Annexes specify that considerations of the molecular structural similarities alone are not sufficient to serve as a justification for grouping different NMs (EU, 2018). That is why ECHA has developed nano-specific guidance for grouping considering properties beyond chemical composition (particle size, shape, or solubility), and highlights the importance of toxicokinetic studies for grouping, read-across, and for in vitro to in vivo extrapolation (ECHA/JRC/RIVM, 2016). New concepts are being developed to improve the integration of mechanistic information, such as the adverse outcome pathways (AOP) concept, or even high-throughput screening (HTS) and omics data (Mech et al., 2019).

Therefore, the EU funded several nanosafety projects, with the aim to provide a conceptual foundation to allow drawing relationships between NMs characteristics and their mode of action regarding toxicity, following a potential uptake. These frameworks and related databases can be used to design tailored NMs with an optimal balance between functionality and hazard or risk [START_REF] Kraegeloh | Implementation of Safeby-Design for Nanomaterial Development and Safe Innovation: Why We Need a Comprehensive Approach[END_REF]. Some of the projects are summarized below.

The FP7 NANoREG project aimed to: i) illustrate the European Commission's definition of nanomaterial; ii) illustrate step-by-step how REACH applies to NMs and identify where the issues still reside; and iii) show the path to solution based on available guidance and work presented in peer-reviewed scientific literature (Gottardo et al., 2017). This project had an important branch towards a common European approach to the regulatory testing of nanomaterials. As a result of this project, a new approach for nano-specific prioritization and risk assessment has been developed and proposed; the steps of this strategy can be found in the work of (Dekkers et al., 2016).

The Horizon 2020 NanoReg2 project, entitled 'Development and implementation of Grouping and Safe-by-Design approaches within regulatory frameworks', intended to develop scientifically based grouping approaches for NMs relevant for regulatory purposes and to establish a Safe(r)-by-Design (SbD) approach for NMs (Mech et al., 2019). This project had a safe innovation approach that combines the SbD concept, applied to industry to include safety considerations from the early stages of a NM development process, and regulatory preparedness, which aims to help regulators to become aware of relevant innovations and to develop the regulatory framework, requirements and methodology accordingly. The NanoReg2 database consolidates earlier experimental data form NANoREG, MARINA, NanoTEST, and Nanogenotox and contains information from more than 6000 experiments regarding physicalchemical properties, (eco)toxicology and environmental fate (Jantunen et al., 2018).

The Horizon 2020 project called GRACIOUS (Grouping, read-across and classification framework for regulatory risk assessment of manufactured nanomaterials and safe(r) design of nano-enabled products) provides a science-based framework that supports the risk assessment of NMs (https://www.h2020gracious.eu/). The objectives of the project are: i) Asses stakeholder needs for grouping and read-across for regulatory risk assessment; ii) To inform about SbD of high-performance products; iii) To identify and test hypotheses, descriptors, criteria and guiding principles by means of ITS composed of relevant experimental and modelling tools; and iv) To integrate them into a framework for grouping, read-across and classification, which will be delivered as a guidance document and a software module.

Nanoecotoxicology

Ecotoxicology is an interdisciplinary field that study the effects of toxic chemical or biological agents on biological organisms at the population, community or ecosystem level. In the last decades, ecotoxicology has developed rapidly along with the industrial pollution of the environment and it has become an important part in environmental and ecological risk assessment [START_REF] Hoffman | Handbook of Ecotoxicology[END_REF].

Ecotoxicological tests were proposed to add toxicity-based criteria to the currently existing policies for the meaningful evaluation of the environmental hazard. Then, with the boom of nanotechnology and NMs being produced and released into the environment, their potential harmful properties on ecosystems had to be evaluated and between 2004 and 2006 scientific publications about NMs started to appear with the word "ecotoxicity" in them [START_REF] Kahru | From ecotoxicology to nanoecotoxicology[END_REF].

NMs interaction with biological systems

The NMs inherent properties, such as particle size, aggregation/dispersion state, ionsrelease capacity, concentration, surface chemistry, and photo-chemical properties, affect the performance of NMs, which may, therefore, influence their toxicity [START_REF] Nguyen | Microalgal ecotoxicity of nanoparticles: An updated review[END_REF]. In turn, the environmental conditions, such as pH, temperature, ionic strength, composition and concentration of natural organic matter; play an important role on NMs behavior and their interaction with organisms [START_REF] Rocha | Ecotoxicological impact of engineered nanomaterials in bivalve molluscs: An overview[END_REF].

Therefore, the understanding of the relationship between the NMs characteristics and behavior in the environment with the NMs toxic effects is fundamental in ecotoxicological studies and environmental risk assessment [START_REF] Corsi | Eco-Interactions of Engineered Nanomaterials in the Marine Environment: Towards an Eco-Design Framework[END_REF]. The study of the correlations between the physicochemical properties of NMs and their toxicity is, currently, in the core of the research direction in nanoecotoxicology with grouping purposes (Hund-Rinke et al., 2018).

Exemplifying the aforementioned, Figure 7 shows that the number of publications in the last 10 years including NMs toxicity and NMs characterization/behavior display a similar trend.

But some authors differ from this concept, arguing that the causal link between particle properties and biological effects is not well established and represents the most eminent quest of nanoecotoxicological investigation (von Moos and Slaveykova, 2014). [START_REF] Corsi | Eco-Interactions of Engineered Nanomaterials in the Marine Environment: Towards an Eco-Design Framework[END_REF].

The interaction between NMs and the aquatic organisms can be chemical or physical.

Although, NMs toxicity towards an organism can be generated by a series of combinations of physical and chemical interactions impacting at molecular, cellular, individual, populational and ecological levels.

To resume the principal concepts of what is known about the interactions and toxicity of NMs towards aquatic organisms, in the next paragraphs, a large scan of information is presented starting from the specific chemical and physical interactions, shading towards the combination of both and focusing on the NMs toxic effects towards some relevant species from the freshwater ecosystems.

Chemical, biochemical and molecular interactions and effects

The chemical impact of NMs in the organisms will be related to their core and coating compositions, as well as their dissolution profiles [START_REF] Misra | The complexity of nanoparticle dissolution and its importance in nanotoxicological studies[END_REF]. Certain NMs, such as TiO2 and ZnO, spontaneously produce ROS at their surface due to their chemical composition and surface properties [START_REF] Li | Mechanism of Photogenerated Reactive Oxygen Species and Correlation with the Antibacterial Properties of Engineered Metal-Oxide Nanoparticles[END_REF] (Fu et al., 2014).

Concerning the biochemical interaction and the effects at the organism's cellular level, the cell wall and the cell membrane are the protective barriers that prevent the entrance of NMs into the cells. But NMs can be internalized by process as endocytosis, phagocytosis and pinocytosis (Huerta-García et al., 2019). Once inside the cell, NMs were found to be lodged in the cytoplasm [START_REF] Werlin | Biomagnification of cadmium selenide quantum dots in a simple experimental microbial food chain[END_REF], mitochondria [START_REF] Iswarya | Trophic transfer potential of two different crystalline phases of TiO2 NPs from Chlorella sp. to Ceriodaphnia dubia[END_REF], algal vacuoles [START_REF] Zhao | Interactions of CuO nanoparticles with the algae Chlorella pyrenoidosa: adhesion, uptake, and toxicity[END_REF], and nucleus [START_REF] Bhuvaneshwari | Dietary transfer of zinc oxide particles from algae (Scenedesmus obliquus) to daphnia (Ceriodaphnia dubia)[END_REF].

NMs interaction with organisms cells can cause cytotoxicity and the most common mechanisms are: oxidation due to increase of ROS, cell membrane damages (perforation), cytoskeleton damages, disturbances in intracellular transport, disturbances in transcription and DNA damages, increasing mutagenesis, mitochondria damages leading to a cell energy imbalance, interference with the lysosomes formation obstructing autophagy and triggering apoptosis [START_REF] Ajdary | Health Concerns of Various Nanoparticles: A Review of Their in Vitro and in Vivo Toxicity[END_REF].

Details of some of these subcellular and molecular effects of NMs, as presented by (Châtel and Mouneyrac, 2017) are summarized as follows.

The ROS induction, caused by NMs, results from pro-oxidant functional groups on their reactive surface, active redox cycling on the surface of NMs due to transition metal-based NMs, and particle-cell interaction. ROS production leads to the induction of several detoxification enzymes as cytochrome P450 (CYP1a) (phase I), gluthatione S-transferase (GST) (phase II), superoxide dismutase (SOD) and catalase (CAT); for which, several studies showed deregulations on the NMs exposed organisms [START_REF] Canesi | Biomarkers in Mytilus galloprovincialis exposed to suspensions of selected nanoparticles (Nano carbon black, C60 fullerene, Nano-TiO2, Nano-SiO2)[END_REF] [START_REF] Buffet | Behavioural and biochemical responses of two marine invertebrates Scrobicularia plana and Hediste diversicolor to copper oxide nanoparticles[END_REF][START_REF] Mouneyrac | Fate and effects of metalbased nanoparticles in two marine invertebrates, the bivalve mollusc Scrobicularia plana and the annelid polychaete Hediste diversicolor[END_REF].

Oxidative stress may also be a consequence of immunological response of organisms following exposure to NMs. Many affectations to the organisms' immune system, caused by NMs have been observed (phagocytosis, cytokines and chemokines production, lysozyme release, etc). These impacts can significantly decrease their ability to defend themselves against pathogens and infectious diseases. NMs related induction in pro-inflammatory and in misfolded protein repair genes are also reported .

Due to their small size, NMs can reach the cell nucleus, and have a direct interaction with the DNA or DNA related proteins damaging the genetic material. Alternatively, DNA damage may arise through indirect mechanisms where the NMs does not physically interact with the DNA molecule, but through other processes such as oxidative stress and immune response activation. This, in turn, leads to cause DNA-strand breaks, mutations, oxidative DNA adducts, and chromosomal fragmentation; followed by programmed cell death or apoptosis. Some NMs have shown possible mutagenic activity as they are able to induce DNA damages, chromosomal aberrations, micronuclei, and aneuploidy [START_REF] Sycheva | Evaluation of the mutagenic properties of nanomaterials[END_REF].

Physical interactions and effects on species

The physical impact can be understood as the mechanical effects, which is not related to the chemical reactions/properties of the NMs [START_REF] Nguyen | Microalgal ecotoxicity of nanoparticles: An updated review[END_REF]. This kind of interactions are considered as a key event, that can determine the NMs uptake and further toxic effects on the organisms [START_REF] Wang | TiO2 nanoparticles in the marine environment: Physical effects responsible for the toxicity on algae Phaeodactylum tricornutum[END_REF].

Examples of these impacts are described as diminution of light access, due to a shading effect, causing nutrient intake limitations [START_REF] Rogers | Physico-chemical behaviour and algal toxicity of nanoparticulate CeO2 in freshwater[END_REF] and cell wall damage derived from cell entrapment [START_REF] Wang | TiO2 nanoparticles in the marine environment: Physical effects responsible for the toxicity on algae Phaeodactylum tricornutum[END_REF]. Other physical impacts of NMs on the cell surfaces of microorganisms, are induced permeability and alterations of the membrane properties, as well as impaired respiration of the cell [START_REF] Morones | The bactericidal effect of silver nanoparticles[END_REF]. Moreover, the physical characteristics of microorganism cells, including adhesiveness and hardness or elasticity, would change due to NMs attachment [START_REF] Zhang | Impacts of Hematite Nanoparticle Exposure on Biomechanical, Adhesive, and Surface Electrical Properties of Escherichia coli Cells[END_REF].

Under some physical stimuli, NMs can convert one physical input into another form of energy as photoactivation that leads to the production of ROS; on the other hand, some NMs as graphene can diminish their toxicity after irradiation due to the production of negative charges that create an electrostatic repulsion towards algae [START_REF] He | Toxicity of engineered nanomaterials mediated by nano-bio-eco interactions[END_REF]. Another example of reduced NMs toxicity by physical interaction is the formation of an eco-corona, which is a layer of NOM that will confer stability of the NM in suspension and that will physically prevent the contact with the organisms, reducing its toxicity.

In Figure 8 the physical interactions of NMs with algae and daphnids are summarized.

Next, a focus of the physical interaction and the main toxic effects of NMs towards specific group of representative organisms of the aquatic ecosystems as algae, microcrustaceans and fish, are described.

• Algae

The reported mechanisms attributing toxicity of NMs to microalgae are agglomeration, adsorption, shading effects and oxidative stress. Accordingly, the physiological changes that have been reported include: severe membrane damage, thickening of the extracellular polymeric substances layer, growth and nitrogen fixation activity inhibition, increase levels of ROS, decrease levels of chlorophyll and carotenoids, inactivation of antioxidant enzymes, lipid peroxidation, opening of intra-thylakoidal spaces and cell structural damages [START_REF] Zhao | Interactions of CuO nanoparticles with the algae Chlorella pyrenoidosa: adhesion, uptake, and toxicity[END_REF]) [START_REF] Saxena | Nanoecotoxicological Reports of Engineered Metal Oxide Nanoparticles on Algae[END_REF].

• Microcrustaceans

Disruptions in daphnids molting processes are reported as physical NMs toxicity, as effect linked to the NMs attachment to the daphnid's carapace (Dabrunz et al., 2011).

Concerning behavioral effects of NMs, microcrustaceans exposed to metal oxide and carbonbased NMs showed reduced swimming speed in a concentration-dependent manner. The main causes are identified as physical interferences of the NMs with the organisms' carapace and physiological manifestations of oxidative stress and other toxic effects [START_REF] Bownik | Daphnia swimming behaviour as a biomarker in toxicity assessment: A review[END_REF].

Another important reported NMs impact in filtering zooplanktonic species is the reduction of food availability (due to NMs aggregation towards algae) which dramatically affects growth and reproduction of the organisms [START_REF] Campos | Effects of nanoparticles of TiO2 on food depletion and life-history responses of Daphnia magna[END_REF].

• Fish

Concerning the physical interactions and associated effects towards fish, as resumed by [START_REF] Handy | Effects of Nanomaterials on the Body Systems of Fishes: An Overview from Target Organ Pathology[END_REF], exposure of fish to NMs can compromise the integrity of external barriers such as the gut and the gills. Injuries include fatty change in the liver, the presence of necrotic cells in the renal tubules, melanomacrophage deposits in the spleen and degeneration of the skeletal muscle fibers. There is also evidence of indirect brain pathology, most likely arising from vascular injury and/or systemic hypoxia. Finally, effects on sense organs and changes in general behavior, due to altered sensory perception; as well as changes in the swimming behavior have been observed [START_REF] Boyle | Subtle alterations in swimming speed distributions of rainbow trout exposed to titanium dioxide nanoparticles are associated with gill rather than brain injury[END_REF].

• Interspecies interactions

The NMs long-term impact on aquatic environments ranges from microbial to ecological scale [START_REF] He | Toxicity of engineered nanomaterials mediated by nano-bio-eco interactions[END_REF]. Trophic transfer of NMs has been verified for many NMs and it has been described as dependent of 4 key factors: the NMs environmental transformations, uptake and accumulation in the prey organism, internal fate and localization in the prey and the digestive physiology of the predator [START_REF] Rosendal Tangaa | Trophic transfer of metal-based nanoparticles in aquatic environments: a review and recommendations for future research focus[END_REF].

Algae and daphnid regulatory toxicity test results for NMs

Currently, the toxicity assessment of NMs is been carried out through the assays from the available standard guidelines for ecotoxicity (Boros and Ostafe, 2020). The OECD Working Party on Manufactured Nanomaterials (WPMN) has concluded that, in principle, the test guidelines are considered to be suitable also for the testing of NMs, although some adaptations were found to be necessary (Hund-Rinke et al., 2016).

Concerning the regulatory algae toxicity test, it is the OECD 201 Freshwater Alga and Cyanobacteria Growth Inhibition Test. This test exposes an exponential growing population to the substance for a period of 72h, and the response is evaluated as a function of the exposure concentration in comparison with the average growth of control cultures (OECD, 2011a).

The EC50 values after exposures of 72h of some of the NMs of interest for this document, are shown in Table 1.

Table 1:

Values of 72h EC50, EC20 and IC50 of some metal oxide NMs on algae. Modified from [START_REF] Nguyen | Microalgal ecotoxicity of nanoparticles: An updated review[END_REF], [START_REF] Wang | TiO2 nanoparticles in the marine environment: Physical effects responsible for the toxicity on algae Phaeodactylum tricornutum[END_REF] and [START_REF] Saxena | Nanoecotoxicological Reports of Engineered Metal Oxide Nanoparticles on Algae[END_REF]. The OECD 202 Daphnia sp. acute immobilisation test Guideline (OECD, 2004) has been used to assess the NMs toxicity towards daphnids (usually Daphnia magna Staus). The organisms are exposed for 48h at a range of at least 5 concentrations of the tested substance.

NMs

The EC50 values of some of the NMs of interest for this document are shown in Table 2. Microalgae are found all over the world. They are mainly distributed in the waters but are also found on the surface of all type of soils. Although they are generally free-living, but a certain number of microalgae can live in symbiotic association with a variety of other organisms [START_REF] Tomaselli | The Microalgal Cell, in: Handbook of Microalgal Culture[END_REF]. Microalgae capture sunlight and perform photosynthesis by producing approximately half of the atmospheric oxygen on earth and absorbing massive amounts of carbon dioxide as a major food source [START_REF] Safi | Morphology, composition, production, processing and applications of Chlorella vulgaris: A review[END_REF]. Microalgae can grow in both fresh and marine water as well as in almost every environmental condition on earth [START_REF] Lee | Phycology[END_REF].

Freshwater microalgae are generally used in ecotoxicological tests because they belong to the first level of the trophic chain, so any disturbance in their dynamics might affect the ecosystem higher levels; they are also very sensitive to changes in their environment and present the advantage of having a short life cycle, allowing the evaluation of toxic effects over several generations [START_REF] Silva | Ecotoxicity tests using the green algae Chlorella vulgaris--A useful tool in hazardous effluents management[END_REF]. Additionally, tests with unicellular organisms, show a greater reproducibility, reliability and robustness than multicellular tests of organisms [START_REF] Shaw | Principles of environmental toxicology[END_REF].

Raphidocelis subcapitata

Raphidocelis subcapitata (Korshikov) was formerly known as Pseudokirchneriella subcapitata or 'Selenastrum capricornutum'. Based on 18S rRNA phylogeny, R. subcapitata belongs to the family Selenastraceae, order Sphaeropleales, but its phylogenetic position in the group has not been resolved [START_REF] Suzuki | Raphidocelis subcapitata (=Pseudokirchneriella subcapitata) provides an insight into genome evolution and environmental adaptations in the Sphaeropleales[END_REF]. This sickle-shaped freshwater green microalga is normally found in unicellular form and its dimension is approximate 40-60 µm 3 .

The diameter of the arc (154 -360°) oscillates between 4,8 and 10,8 μm, and the width ratio R. subcapitata cell cycle consist of 3 phases: growth of the mother cell, cell division with two nuclear divisions forming four daughter cells (autospores), and release of the autospores through rupture of the parental cell wall without forming colonies [START_REF] Machado | Modification of cell volume and proliferative capacity of Pseudokirchneriella subcapitata cells exposed to metal stress[END_REF]. This exponentially high growth rate allows to culture large quantities in limited periods of time.

Currently, R. subcapitata is the best known and most frequently used species of ecotoxicological bioindicator because of its high growth rate and sensitivity to toxicants. Hence it is considered one of the most important organisms in bioassays of water quality, algicides and environmental assessments [START_REF] Yamagishi | Cell reproductive patterns in the green alga Pseudokirchneriella subcapitata (=Selenastrum capricornutum) and their variations under exposure to the typical toxicants potassium dichromate and 3,5-DCP[END_REF].

Desmodesmus subspicatus

Desmodesmus subspicatus (Chodat, 1926), formerly known as Scenedesmus subspicatus, is a freshwater microalga that belongs to the genus Desmodesmus in the family Scenedesmaceae of Chlorophyceae microalgae. This genus is characterized by spheroid to ellipsoid cells, with regular chains of 4 to 8 cells (Figure 10). It differentiates from the genus Scenedesmus, from the same family, due to their spines. These microalgae naturally occur in fresh continental water, especially in nutrient-rich environments. They are characterized by a high degree of flexibility allowing them to be true cosmopolitans and to withstand harsh conditions. Although Desmodesmus are usually thought of as four celled colonies, many species may produce unicells as well. These unicells may result when the daughter cells fail to join together and additional spines may appear on each cell, they may also be the product of colony disintegration. [START_REF] Silva | Heterotrophic growth of green microalgae Desmodesmus subspicatus in ethanol distillation wastewater (vinasse) and lipid extraction with supercritical CO2[END_REF] [START_REF] Lürling | Phenotypic plasticity in the green algae Desmodesmus and Scenedesmus with special reference to the induction of defensive morphology[END_REF]. Members of this genus are used for biodiesel production and for wastewater treatment [START_REF] Xin | Growth and lipid accumulation properties of a freshwater microalga Scenedesmus sp. under different cultivation temperature[END_REF]. And along with R. subcapitata, D. subspicatus is a recommended species for the ecotoxicology test OCDE 201 (OECD, 2011a).

Chlorella vulgaris

The name Chlorella comes from the Greek word chloros (Χλωρός), which means green, and the Latin suffix ella referring to its microscopic size. It is a unicellular microalga that grows in fresh water and has been present on earth since the pre-Cambrian period 2.5 billion years ago [START_REF] Ditfurth | Im Anfang war der Wasserstoff[END_REF] and since then, its genetic integrity has remained constant [START_REF] Safi | Morphology, composition, production, processing and applications of Chlorella vulgaris: A review[END_REF].

The genus Chlorella is formed by cosmopolitan organisms with small, unicellular, ovoidal nonmotile cells that multiplies by autosporulation which is the most common asexual reproduction in algae. It grows in autotrophic, heterotrophic and mixotrophic conditions [START_REF] Tomaselli | The Microalgal Cell, in: Handbook of Microalgal Culture[END_REF].

Martinus Willem Beijerinck, a Dutch researcher, first discovered Chlorella vulgaris it in 1890 as the first microalga with a well-defined nucleus [START_REF] Beijerinck | Culturversuche mit Zoochlorellen, Lichenengonidien und anderen niederen[END_REF]. C. vulgaris is a spherical microscopic cell with 2-10 μm diameter. At the mature stage, cell wall thickness can reach 17-21 nm and it is composed by a microfibrillar (chitosan-like) layer of glucosamine which accounts for its rigidity. Nevertheless, cell wall thickness and composition are not constant because they can change according to different growth and environmental conditions.

This species has a single chloroplast with a double enveloping membrane composed of phospholipids; inside the chloroplast, starch granules of amylose and amylopectin can be formed. The chloroplast also stores a cluster of fused thylakoids where chlorophyll is synthesized masking the color of other pigments such as lutein. The pyrenoid contains high levels of ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO) and is the center of carbon dioxide fixation [START_REF] Safi | Morphology, composition, production, processing and applications of Chlorella vulgaris: A review[END_REF]. The morphology of Chlorella vulgaris is presented in Figure 11. C. vulgaris is a non-motile reproductive cell (autospore) that reproduces asexually and rapidly (Figure 12). Within 24 h, the mother cell will form four daughter cells with their own cell wall. After their maturation, the mother cell wall ruptures, allowing the liberation of the daughter cells and the remaining debris of the mother cell are consumed as food [START_REF] Yamamoto | Regeneration and maturation of daughter cell walls in the autospore-forming green alga Chlorella vulgaris (Chlorophyta, Trebouxiophyceae)[END_REF]. Chlorella vulgaris is one of the most important species in the microalgal industry; it is cultivated and sold for many purposes: as food due to its high content of protein (>55% of dry weight), as mass culture for CO2 reduction, as medical treatments due to its immune-modulating and anti-cancer properties, and even as biodiesel producer due to its capacity of accumulating important amounts of lipids [START_REF] Safi | Morphology, composition, production, processing and applications of Chlorella vulgaris: A review[END_REF].

Freshwater microcrustaceans

Freshwater microcrustaceans refers mainly of two groups of organisms: Cladocera and

Copepoda [START_REF] Novichkova | The first data on the freshwater microcrustaceans of Shokalsky Island (Russian Arctic)[END_REF]. They constitute a dominant component of limnological and zooplankton systems, they are the major food source for many freshwater organisms like fish and other crustaceans and play an important role in the food chain and energy transfer in the ecosystem [START_REF] Karuthapandi | Cladoceran Diversity, Distribution and Ecological Significance[END_REF]. Cladoceran organisms are specially known to be good laboratory test species due to their short life cycles and ease of culturing [START_REF] Wogram | Regulatory challenges for the potential use of ecological models in risk assessments of plant protection products. Ecological models for regulatory risk assessments of pesticides: developing a strategy for the future[END_REF].

Daphnia magna

Daphnia magna (Straus, 1820), is a freshwater microcrustacean, commonly known as waterflea, due to the jumping-like behavior they exhibit while swimming. It belongs to Kingdom: Animalia, Phylum: Arthropoda, Subphylum: Crustacea, Class: Branchiopoda, Order:

Cladocera, Family: Daphniidae. Like many aquatic invertebrates, daphnia occupies an important place in the food chains of aquatic ecosystems. A consumer of phytoplankton algae and other small organisms, it in turn can be the prey of fish and many carnivorous aquatic animals [START_REF] Parisot | Étude mécaniste des effets transgénérationnels des radiations ionisantes alpha et gamma chez Daphnia magna[END_REF]. This species is one of the most investigated freshwater organisms worldwide and it is widely used as a model organism in ecological, ecotoxicological and ecosystem biomonitoring research (C.S. [START_REF] Reynolds | Daphnia: Development of Model Organism in Ecology and Evolution -2011[END_REF][START_REF] Bojadzija Savic | Metabolic interactions between Daphnia and cyanobacteria[END_REF].

Daphnia magna lives mainly in stagnant bodies of water such as lakes, ponds or temporary pools. Its geographic distribution stretches from the eastern United States to the north of western Europe. Although daphnia can live in environments where water quality varies greatly with the seasons, this species prefers relatively temperate temperatures (18-22 ° C) and neutral waters (pH 7.2 -8.5) [START_REF] Parisot | Étude mécaniste des effets transgénérationnels des radiations ionisantes alpha et gamma chez Daphnia magna[END_REF]. Daphnia contributes to the ecological balance of pelagic areas by naturally purifying water, participating in the mixing of the water column, regulating phytoplankton population and transferring energy and carbon to higher tropic levels because they are a preferred prey of many aquatic organisms [START_REF] Bojadzija Savic | Metabolic interactions between Daphnia and cyanobacteria[END_REF].

The anatomy (Figure 13), physiology and life cycle of D. magna have been described

in detail by [START_REF] Ebert | Introduction to Daphnia Biology, Ecology, Epidemiology, and Evolution of Parasitism in Daphnia[END_REF]) and it will be presented next in a summarized form. D. magna is described having a body size from 0.5 to 6 mm, that is enclosed by an often transparent and uncalcified carapace, made of polysaccharide chitin. This carapace has a double wall, between which hemolymph flows. Daphnia has two sets of antennae, to sense out their surroundings, maxilla, mandibles and limbs acting as a respiratory and feeding system, as well as a pair of claws at the end of the abdomen. Males are distinguished from females by their smaller size, larger antennules, modified post-abdomen, and first legs, which are armed with a hook used in clasping. The carapace of juveniles, males and females is terminated by a caudal spine.

The nervous system is characterized by the cerebral ganglion, which is located close to the gut and near the eye. Juvenile and adult organisms have one large compound eye, that helps them to orient while swimming, although embryos show two brownish eye spots that fuse during the last part of the development. D. magna has an open blood circulation. The heart is located dorsally and anterior from the brood chamber and it beats 200 times per minute at 20°C.

In adverse conditions, Daphnia can develop extracellular respiratory protein hemoglobin (Hb)

to support oxygen transport and to increase oxygen uptake from the water. They absorb ions with chloride-absorbing glands and as for other freshwater animals, osmoregulation may be a problem (too few solutes).

D. magna is a filter feeder, it feeds on small, suspended particles in the water. Green microalgae are the preferred nourishment. The food is gathered using a filtering apparatus, consisting of phylopods, which are flattened leaf-like legs that produce a water current that flows anterior to posterior. The collected particles are then, transferred into the food groove by special setae and they pass through the gut by peristaltic contractions. Their tubular gut has three parts: esophagus, midgut (containing microvilli), and hindgut. Two small digestive ceca (diverticula) can be seen in the head section of the midgut. Food is expelled from the hindgut by peristaltic movement but also requires the pressure of more recently acquired food particles.

The dynamics of food uptake answer to a functional response type 1 (see Figure 14). Below a certain food concentration, the feeding rate is proportional to the food concentration, and the filtering rate is maximal. Above this level, the feeding rate is constant because the filtering rate decreases with increasing food concentration in the water. The life cycle of D. magna is characterized by two modes of reproduction: asexual and sexual, as showed in Figure 15. Asexual reproduction occurs in favorable environmental living conditions. During these periods, the populations are mainly composed by females. Without any other sex fertilization, adult females produce genetically identical eggs. This is called "parthenogenetic" reproduction.

The first egg laying of an adult female occurs after 5 to 10 days of life, when she has reached maturity. The eggs are deposited in the brood chamber of the females, located dorsally beneath the carapace. The embryos hatch after one day but remain in the brood chamber, in order to complete their development. After 3-4 days, the mother daphnia release the juveniles by ventral flexion of the post-abdomen. The neonates will go through 4 to 6 different stages of development, called "instars" and they correspond to the periods between molting.

Under optimal conditions, the life cycle of D. magna is divided into three different stages: embryonic (3 days), juvenile (6 days) and adult (until death). At 20 ° C, an adult daphnia releases its first clutch after 9 days and new eggs are successively released every 2-3 days until their death. These cyclic spawning occur after each molt event. The clutch size varies from 10 to 40 eggs depending on the age of the mother. In optimal conditions, females can live up to two months. Parthenogenetic generations will follow each other while the environmental conditions remain favorable.

Sexual reproduction occurs when environmental living conditions are unfavorable to them (lack of food, increased competition, lowered temperature, decreased day length, chemical stress, etc.). During these periods, the populations of D. magna give birth to a bisexual generation composed of males and females. Females are fertilized by males and produce two haploid eggs from each ovary. These eggs are encapsulated in a protective structure that allows the eggs to withstand adverse conditions, called "ephippium", and the whole is called ephippia.

Ephippia is released during periods of molting and it sinks to the bottom of the water column or floats with the help of small gas chambers; it can also be transported by the wind, animals or water streams. The hatching of these resting eggs is only induced by external stimuli, when environmental conditions are favorable again. The individuals resulting from these eggs will constitute a population genetically different from the mother daphnia. Additionally, they can be observed clinging occasionally to plants or other substrates to pick up small particles when food is lacking.

Nanomaterials of the study

Selection of NMs

For the present study, a set of NMs was selected based on different criteria : the set would be formed by a group of well-known NMs from which the main characteristics were already widely studied; the selected NMs would have different physicochemical characteristics and the set would be formed by NMs that can form suspensions in water or biological media for at least 48h.

The selected group of NMs that meet the aforementioned characteristics is composed by 10 NMs that come from the Joint Research Center (JRC) Nanomaterial Repository, from which:

-Six are composed by TiO2 (NM100, NM101, NM102, NM103, NM104 and NM105).

These NMs have different characteristics as crystalline phase (anatase, rutile), coatings, zeta potentials, hydrophobicity, etc.

-One composed by ZnO (NM110), hexagonal non coated NMs.

-Three SiO2 NMs amorphous, generally negatively charged.

Additionally, there are 2 CeO2 NMs. One is the denominated CeO2<10nm from NanoByk ® , coated with tri-ammonium citrate. The other one is CeO2<25nm from Sigma Aldrich. The denomination came from the pristine size of the NMs.

The selected NMs are presented in (Zhang et al., 2015) and (Manier et al., 2013).

Preparation of NMs suspensions

Stock suspensions for all* NMs were prepared at 2.56 g/L following the dispersion protocol from the NANOGENOTOX Program [START_REF] Keld | The NANOGENOTOX dispersion protocol for NANoREG[END_REF], but without using the bovine serum albumin nor the ethanol. This because it has been observed that these substances can dramatically alter the characteristics and behavior of NMs through a protein corona on the NMs and thereby impact the toxicity observed (OECD, 2020). Suspensions of 15 mL were prepared using ultrapure water and 38.4 mg of NMs powder. First, a phase of prewetting was carried out to hydrate the powder using some drops of ultrapure water and revolving gently. Then, the rest of the water volume was added, and the suspension was sonicated with a Vibra-CellTM (VCX 750, Sonics) sonicator during 16 min at 20% amplitude in an ice bath, delivering approximatively 7056 ± 103 J or a total delivered acoustic power of 7.35 ± 0.05 Watt [START_REF] Booth | NANoREG D4.12 SOP Probe Sonicator Calibration for ecotoxicological testing[END_REF]. Photos of the suspensions are presented in Figure 16.

* Excepting for CeO2<10nm that was provided already in aqueous suspension at 217.8 g/L. This NM was only diluted in ultrapure water to arrive at the stock suspension concentration of 2.56 g/L and it was not sonicated. From here on, the handling was the same as the other NMs. (Photos: Rivero Arze, A.).

NMs attachment to microalgae experiments

Algae cultures

All algae were bought at the Culture Collection of Algae and Protozoa (CCAP), https://www.ccap.ac.uk. The algae arrived in a suspension form that was conserved refrigerated (4°C) and the inoculums were made as soon as possible. For the inoculum, a LC media was prepared (composition in Annex 1), and the algae suspension added to 100 mL of media in a 250 mL Erlenmeyer at a proportion of algae/media of 1/10 v/v following the provider specifications. The inoculum was let to grow for 1 week in an incubator at 22°C ± 3°C with white horticultural light (5500-6000 lux) in orbital agitation (100-110 rpm) until they were fully green. Then, they were divided in several aliquots of 15 mL each (Fig. 17.A.) and stored at 4°C.

These aliquots can be used upon 2 years after prepared.

To start a continuous culture called "mother strain", one aliquot at a time was used. The aliquot was taken from the refrigerator and let take room temperature. Algae is found at the bottom of the tube due to sedimentation. The supernatant media was discarded and the bottom down milliliters containing the sedimented algae were inoculated in fresh LC media. The mother strain was left to grow for 1 week in the upper explained conditions, until it was fully green (Fig. 17.B. and 17.C.). At the end of the week the mother strain was renewed into a fresh media. This operation was done up to 10 weeks starting from 1 aliquot, after this period, a new aliquot was used.

The algae culture used in the algae-NMs experiments is called "pre-culture". The precultured algae are preferable used in experiments because all of them are in the same stage of development (exponential growth). Inoculums* for the preculture were made starting from a mother strain culture in 100 mL of OECD media (composition in Annex 1). They were placed in an incubator for 3 days in orbital agitation (Fig. 17 *The volume of the inoculum for pre-cultures depended on the algae species:

-For Raphidocelis subcapitata: 1 mL.

-For Desmodesmus subspicatus: 5 mL.

-For Chlorella vulgaris: 3 mL.

For all the algae cultures (aliquots, mother cultures and pre-cultures), each of the Erlenmeyer flasks had a plug that allows the air exchange. The plugs were BIO-SILICO ® or a cotton plug, made at the laboratory, with carded cotton inside a piece of gauze. For the experiments, an adequate concentration of pre-culture algae was needed (see point 2.3. of this chapter for details). The concentration was found to be 5*10 5 cells/mL, and in order to arrive at this concentration in the flow cytometry sample, a working algae concentration of 1*10 6 cells/mL was needed. This concentration was achieved diluting the pre-culture algae suspension, which concentration was found counting the cells under the microscope in a BRAND™ Malassez counting chamber.

Development of NMs-algae short time contact test

In order to obtain an homogenic contact between the algae and the NMs a short-time contact test was developed. This setup prevented algae or NMs sedimentation and ensured that they both were in contact all along the experiment. The importance of the short period of time for this exposure is to ensure that the measures that will be done are mainly from the attachment and to maintain the number of cells before natural multiplication or diminish from possible NMs toxic effect.

The short-time contact test was developed using an equipment constructed locally with a capacity of 60 places for glass vials, that maintain a constant temperate through a system of cycling water. A horticultural lightening was installed to provide light for long tests, and a plate of 60 magnetic stirring spots was placed in the bottom, so each sample can be stirred individually. A 1,5 cm magnetic stirring rod was placed inside each glass vial and the magnetic agitation was set to 300 rpm. Photos of the equipment are presented in Figure 18. The experiment tested 10 different concentrations of NMs (0.1; 0.5; 1; 2.5; 5; 7.5; 10; 12.5; 25 and 50 mg/L) for each of the 3 algae species. The setup of the vials preparations volumes is detailed in Table 4.

Sample

Flow cytometry analysis

The basic principle of flow cytometry is the passage of cells in single file in front of a laser so they can be detected, counted and sorted. Cell components are normally fluorescently labelled and then excited by the laser to emit light at varying wavelengths (IITK, 2018). It offers several unique advantages as it allows fast, relatively quantitative, multiparametric analysis of cell populations at the single cell level. In addition, it also enables physical sorting of the cells to separate the subpopulations based on different parameters [START_REF] Manohar | Flow cytometry: principles, applications and recent advances[END_REF].

The flow cytometer is comprised of three key systems: the fluidic, the optical and electronic system (see Figure 20). In the flow cytometry analysis of algae-NMs interaction, the analyzed parameters were fluorescence (FL1) in the X axe and side scatter logarithm (SSlog) in the Y axe. The measured fluorescence corresponds to the natural fluorescence of the green microalgae, no dye was used.

The SSlog measures the granularity/complexity of the cells. The principle of this analysis is based in the phenomenon that when algae interact with NMs, these lasts will attach around the algae cells making them more complex than the control cells. Heteroagglomerates of algae-NMs can also be formed, which will likewise be more complex than a control cell.

For the analysis of the cytograms, the samples of a non-contaminated algae population were analyzed to find an ideal concentration that does not overcharge the system but that provides enough counted points for the analysis. After some preliminary tests, the chosen concentration for the analysis was 5*10 5 cells/mL. Based on these algae control cytograms the control population profile is determined creating an area that englobes at least 95% of events.

The number of events appearing inside this area for the 3 algae control population samples were averaged and that number was used to compare with each number of dots inside the area for the interaction samples.

As the formation of heteroagglomerates (registered with higher complexity) will diminish the number of events registered by the flow cytometer for a constant algae concentration in the samples, the number of events inside the area were considered to calculate the percentage, and it was not considered the percentage already given by the software that is related to the number of events in the sample.

An internal control (algae + NMs at 5 mg/L) was also analyzed at the beginning, at the middle and at the end of each plate sequence, to verify that there were no deviations in the measures of the cytometer. Finally, a last non contaminated algae sample was analyzed at the end of each pate sequence to verify the cell counts (see plate setup on Figure 19).

The detail of the steps for the analysis of one sample is explained below and showed in Figure 21.

-First, delimit the area of the NMs control and verify that at least 95% of the registered events are counted. In Fig. 21, 99.22% of the events are inside the delimited area -Second, delimit the area of the algae control population and verify that at least 95% of the registered events are counted. In Fig. 21, there are 14846 events inside the delimited area, that represents 99.15% of the total events. Pass the 3 samples to get a mean of the control algae population (CAP). In this case, the mean was found to be 14721.

-Third, create a new dot plot that regroups both, control NMs and control algae delimited areas, and analyze an interaction sample (IS). The number of events inside the algae control is 1385. The percentage of modified algae (%MA) is found according to the following equation:

% 𝑀𝐴 = ( 1 - 𝐼𝑆 𝐶𝐴𝑃 ) * 100
To end, the result for this sample will be 90,59% of modified algae.

Hydrophobicity of the algae-NMs interaction

A team from the Consumer Products Safety Unit at the JRC has developed a method to determine quantitatively the NMs hydrophobicity. The quantification is obtained by comparing the nanomaterial binding affinity to two or more engineered collectors, via analysis of their adsorption kinetics. The adsorption kinetics is calculated by measuring the number of nanoparticles binding to the different collector as a function of time, by Dark-Field microscopy [START_REF] Valsesia | Direct quantification of nanoparticle surface hydrophobicity[END_REF].

A collaboration between the JRC team and INERIS has been established with the objective of determining the hydrophobicity of the NMs in the algae media and in contact with the algae. The test for the algae-NMs interaction used mainly 3 collectors: a hydrophobic collector called T0 which surface has been coated with a plasma deposited layer of polytetrafluoroethylene (PTFE), then two other collectors, called T3 and T4, that are hydrophilic due to several layers of polyelectrolytes deposited on top of the PTFE surface. The polyelectrolyte layers will give a charge to the collector's surface, being T3 positively charged and T4 negatively charged.

Interaction between 5 NMs (TiO2-NM101, TiO2-NM103, TiO2-NM104, SiO2-NM200

and ZnO-NM100) at 10mg/L and 2 algae species (R. subcapitata and D. subspicatus) were tested. Each algae-NMs combination passed through a micro-channel on each one of the 3 collectors so the biding affinity to the surface can be determined.

Uptake and elimination experiments

Preparation of the exposition population

Previous to the start of the experiment, the INERIS daphnids culture was enlarged to be capable to produce more than 1000 neonates per day. This led to the maintenance of a dozen aquariums (2 liters each) containing 40 mothers each (see Figure 22). The media used for raising these daphnids was the Elendt M4 media (composition in Annex 1). Temperature was at 20 ± 2°C with a day-night cycle of 16h of light (1000-1500 lux). They were fed daily with freshwater microalgae (Chlorella vulgaris, Desmodesmus subspicatus and Raphidocellis subcapitata) at 2 mg/L TOC and the offspring was daily removed.

One day before the collection of the daphnids newborns, all the mothers' aquariums were filtered to ensure that all the collected individuals next day will be less of 24h old. The day of the collection, 1100 neonates were placed in 4L aquariums with Elendt M4 media and the same environmental conditions as the mothers but in the experimental chamber to acclimate if any condition was to be different. They grew up synchronically for 7 days in these conditions, arriving to the experiment with 8 days of life.

Experimental design

When the organisms arrived at their 8th day, the accumulation phase started. Prior to the exposure, the daphnids were fed one last time. After one hour they were placed in clean ISO media, where they remained for another hour, to acclimate and to clean their guts. Then, they were placed by groups of 50 individuals in 1L containers with ISO media (composition in Annex 1) spiked with NMs at a concentration of 1mg/L. This concentration was chosen based on the results of [START_REF] Zhu | Toxicity and bioaccumulation of TiO2 nanoparticle aggregates in Daphnia magna[END_REF] work, where a good delimitation of the accumulation and elimination curve had been obtained.

In total 21 containers were put in place for the experiment, 4 replicates of 5 groups for the uptake and elimination phases and 1 control non-contaminated container. This emplacement was used for each one of the NMs and each experiment lasted 1 week in total, making the duration of the complete experiment 12 weeks long (the experimental design is shown in Figure 23). From the 4 replicates of each group 3 were used to obtain the samples for the metal dosing analysis and 1 for the transcriptomic analysis. The groups represent the sampling time-points.

Having 11 sampling points in total, there were distributed as follows: i) the TA0 point was sampled before exposition taking the organisms right from the container were they emptied their guts; ii) the 5 uptake time points were sampled taking 25 individuals from each container from each one of the 5 groups of containers at 1, 2, 4, 6 and 24h; and iii) for the elimination phase the resting 25 individuals from all the 5 groups were sampled at 2, 6, 24, 48 and 120h. After the 24h of uptake phase where no food was provided (including the control group), the daphnids were recovered and placed in containers with clean ISO media. The control container was replaced with another liter of media so the daphnids are under the same conditions. During the elimination phase the organisms were fed daily with R. subcapitata 1.7 mg/L TOC.

Individual's mortality throughout the experiment was registered. Water samples (5 mL)

were also taken to follow the NMs concentration in the media. Water sampling was carried out in triplicates at 0 and 24 hours of uptake, as well as, 0 and 120 hours of elimination.

Samples of daphnids for the ICP-OES analysis were dried at the oven at 70°C for 24h, then they were weighted and stored at -4°C waiting for the analysis. The samples for the transcriptomic analysis were immediately frozen at -80°C waiting to be analyzed. Photos of the sampling and experiment disposition are showed in Figure 24.

Acid digestion

Acids are the primary reagents used for breaking down metal containing compounds.

Microwave metal digestion was developed to maximize analyte retention and yields complete matrix decomposition with little-to-no loss of sample content. Mixture of acids as nitric acid (HNO3) and hydrofluoric acid (HF) can dissolve even the toughest matrices including silica.

(ColdBlock Technologies, 2020).

Samples of TiO2, ZnO and CeO2 NMs exposed daphnids and media were acid digested in 50 mL Teflon reactors using 3 mL of ultra-pure HNO3 and 2 mL of fluoroboric acid (HBF4).

For the SiO2 NMs samples HF was used instead of HBF4 and they were neutralized with 20 mL of 55 g/L ultra-pure boric acid after digestion.

The acid digestion was carried out using a microwave (Mars6, CEM) with a capacity of 40 Teflon reactors. Samples were processed by groups of 39 samples and 1 method blank per group. For the SiO2 NMs samples, the process consisted on a 30 min temperature rise ramp until 205°C and hold for 30 min (1800 W). For the rest of the samples the program had four temperature rise ramps, i) to 120°C for 15 min, ii) to 140°C for 5 min, iii) to 160°C for 5 min, iv) to 200°C for 10 min with a hold at that temperature for 20 min. All samples were adjusted to a final volume of 50 mL.

ICP-OES analysis

Inductively Coupled Plasma Optical Emission Spectrophotometry (ICP-OES) is now one of the most versatile methods of inorganic analysis. ICP-OES analysis consists in introducing a liquid sample through a nebulizer which transforms it, into an aerosol composed of microdroplets. The sample arrives to an argon plasma torch which has high electron density and temperature (10000K), and this energy is used in the excitation-emission of the sample.

When the excited atoms return to low energy position, emission rays (spectrum rays) are released and the emission rays that correspond to the photon wavelength are measured. The element type is determined based on the position of the photon rays, and the content of each element is determined based on the rays' intensity (Hitachi High-Tech GLOBAL, 2001). The schematic operating mode of an ICP-OES is showed in Figure 25. analyzed as quality control of the process. The method blank sample of each acid digestion group was also analyzed, to verify the absence of metal-traces in the reactors. During the analysis run, a control sample from the standard range was analyzed every 10 samples to verify that no curve deviation occurred. Finally, at the end of each run, the low concentration points of the standard range were also verified.

From the standard range concentration curve, the Limit of Quantification (LQ) for each element was found. In Table 5 are presented the LQ for the organisms and the water samples.

Water samples were diluted 10 times for the analysis and a mean weigh of the organisms' sample of 3 mg was used for the LQ of the body burden calculation. For the organisms' LQ calculation a mean weight of daphnids of 3 mg was assumed and water samples were diluted 10 times for the analysis.

Photos of the ICP-OES analysis of the samples are showed below in Figure 26.

Transcriptomic analysis

The real time polymerase chain reaction is considered the most powerful tool for quantitative nucleic acids analysis [START_REF] Bustin | Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays[END_REF]. This new technique is a refinement of the original Polymerase Chain Reaction (PCR) developed by Kary Mullis and coworkers that earn him a Nobel Prize. The principle consists essentially in that any nucleic acid sequence present in a complex sample can be amplified in a cyclic process to generate a large number of identical copies that can readily be analyzed. [START_REF] Kubista | The real-time polymerase chain reaction[END_REF].

Real time quantitative PCR (RT-qPCR) is the most versatile method for single cell mRNA analysis and it also offers quantitative information about transcript levels [START_REF] Bengtsson | Gene expression profiling in single cells from the pancreatic islets of Langerhans reveals lognormal distribution of mRNA levels[END_REF]. The two most commonly used methods to analyze data from RT-qPCR experiments are absolute quantification and relative quantification. Absolute quantification determines the number of copies of a gene relating the PCR signal to a standard curve. The relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control; and the 2 (-𝛥𝛥𝐶𝑡) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments (Livak and Schmittgen, 2001).

All the transcriptomic analysis protocol details are presented in Chapter 5.

Statistical analysis

The statistical analysis of the flow cytometry results, the uptake and elimination results and the transcriptomic results are detailed within the paper of their respective chapters. The same for the MOSAICbioacc model that is available at http://lbbe-shiny.univ-lyon1.fr/mosaicbioacc/, and which principles are detailed within Chapter 4.

Principal component analysis of a data matrix extracts the dominant patterns in the matrix in terms of a complementary set of score and loading plots. It is the responsibility of the data analyst to formulate the scientific issue [START_REF] Wold | Chemometrics and Intelligent Laboratory Systems[END_REF]. This analysis was used to determine NMs clusters of similar characteristics including the NMs pristine properties, their physicochemical characteristics in the different exposure media and all the nano-bio interaction results of algae attachment, uptake and elimination on daphnids as well as transcriptomic analyzed early molecular effects.

Chapter 3: Interaction between freshwater microalgae and nanomaterials 1. Introduction and context

Freshwater microalgae are the first level in the food chain of these ecosystems. When

NMs are released into the water, these organisms are among the most impacted. Physical, chemical and biological effects are described by the literature. The main adverse effects are the diminution to nutrients and light (shading effect), cell wall damage, structural damage, oxidative stress and cytotoxicity [START_REF] Nguyen | Microalgal ecotoxicity of nanoparticles: An updated review[END_REF]. The importance of studying the interaction between NMs and microalgae relies on the importance of microalgae itself, as primary producers in the ecosystem, they constitute the entry way of pollutants in the trophic chain.

In the present study we refer to the first physical encounter between the microalgae cell and the NMs as a first and key event that will determine the chain of effects and interactions that can happen subsequently. This key event is determined by the 'particulate' nature of the NMs which makes it different than the exposition of microalgae to an ionic solution of the same material.

The flow cytometry analysis presents itself as a possibility for the analysis of the algae-NMs interaction due to the capacity of discriminate populations by their complexity. The natural green fluorescence of green algae makes them capable of been studied by this method.

A short-contact test has been developed to assure a homogenic encounter between the algae cells and the NMs.

NMs are currently being evaluated, as other chemical compounds, through the OECD 201 freshwater algae and cyanobacteria growth inhibition test (OECD, 2011a). Some questions have been formulated for the suitability of these tests to analyze the nano-related toxic effect of NMs. Additionally, the analysis of NMs interactions with algae as a ecotoxicity prediction method and as a NMs grouping parameter are of current interest (Hund-Rinke et al., 2020).

The main questions raised when assessing this issue are as following:

• Does the developed short-time contact test and analysis by flow cytometry allow to characterize the algae-NMs interactions?

• Under the same circumstances, do all the studied microalgae species interact in the same way with a given NM?

• Under the same circumstances, do all the studied NMs interact in the same way with a given microalgae species?

• Are there any algal or NMs physicochemical properties that can influence this interaction?

• Does the algae-NMs interaction evolve with the pass of time?

• How the findings about the algae-NMs interaction can be relevant for risk assessment purposes?

Hereby it is presented a conceptual schema that summarizes the scope of this chapter.

Characterization of the nano-bio interaction between metallic oxide nanomaterials and freshwater microalgae using flow cytometry (published)

3. Supplementary results

Kinetics study supplementary results

To determine if a significant difference in the algae-NMs interaction exists between the 15 min, 1 hour and 2 hours exposure, the population coefficient of variation was calculated. In the following table, the counts by flow cytometry of the population of R. subcapitata are presented and the calculation of the coefficient of variation was obtained according to:

((standard deviation / mean) *100) for each population. The highest value is chosen in order to determine the minimal percentage from which a difference is taken as valid. The threshold chosen is 10%. Differences between the percentage of modified granularity at 15 minutes, 1 hour and 2 hours exposure of R. subcapitata for the tested nanomaterials are showed in Table 7.

Statistically different values are distinguished by a "*". Cells in blue show the differences that are statistically different and above 10% (stablished as a threshold that is greater than the coefficient of variation).

Hydrophobicity study of the algae-NMs interaction

A collaboration between INERIS and JRC was stablished, based on the recently JRC method of NMs hydrophobicity determination, to study the attachment between the hydrophobic/hydrophilic NMs with microalgae cell walls. In this context, an experiment of interaction between 5 NMs (TiO2-NM101, TiO2-NM103, TiO2-NM104, SiO2-NM200 and ZnO-NM100) at 10mg/L and 2 algae species (R. subcapitata and D. subspicatus) was carried out.

The final results are still being analyzed by the JRC team, but early visual results seem to confirm the suitability of this method for the algae-NMs interaction as it can be shown on Figure 28. It is important to note that the dark field microscope is focused right on the surface of the collector, consequently the well-focused dots mean that the material is the surface level.

Any other blurred dot showed material floating above the surface, hence not attached. The tested NM103, that is hydrophobic, showed an affinity for the T0 hydrophobic collector (brilliant dots) and the algae can be distinctively seen attached to the collector surface. On the other hand, NM101, that is not hydrophobic, is not attached to the T0 collector (blurred dots)

and no algae can be seen on focus of the microscope.

Algae-NMs interaction TEM study

From the experiment of the hydrophobicity determination, some samples were taken for a Transmission Electron Microscope (TEM) analysis. A total of 10 samples were taken, these samples include 2 algae species: R. subcapitata and D. subspicatus and 5 NMs (TiO2-NM101, TiO2-NM103, TiO2-NM104, SiO2-NM200 and ZnO-NM100). Some exemplifying TEM images, from this systematic study, are presented below as photo plates in Figures 30 and31.

An additional case was studied with the addition of Natural Organic Matter (NOM) to analyze how the presence of NOM can affect the algae-NMs interaction in a context of exposition in natural environments. Since the flow cytometry analysis of this case couldn't be done, this part of the TEM study becomes less relevant for this document. Nevertheless, some images are included, as an indication, in Annex 2. The results of the interaction between R. subcapitata and the tested NMs, showed mainly attachment to the cells, cell covering and some cell wall damage. The interaction keeps being external as for all the studied interactions, no NMs internalization was found. The images confirm, for the most part, the results found by flow cytometry. The similarities and differences are detailed case-by-case below, along with interpretation of the effects showed by the TEM images.

The interaction with the TiO2-NM101 showed algae with some NMs agglomerates around, but not totally surrounded. The most relevant phenomenon observable is the retraction of the algae cell wall in the areas of contact, as shown on Fig. 30.A. and C. The Fig. 30.B. showed a large NMs agglomerate in contact with one alga, partially recovering the surface of the cell.

The TiO2-NM103 interaction showed more invasive effects towards the algae cell walls. For the interaction between D. subspicatus, the description and analysis of the images are as follows:

TiO2-NM101 showed very little agglomerates around the cells. In Fig. 31.C. some NMs can be seen near the cell wall but margin of distance between both of them exists.

For the interaction with TiO2-NM103 and TiO2-NM104, no big agglomerates are seen in contact with the cells, only few little groups of NMs. In Figure 31.F. a group of NMs is showed attached to the cell wall and the composition analysis has confirmed that it is titanium.

Similar to the case of interaction with R. subcapitata, the ZnO-NM110 showed almost no visible ZnO particle agglomerates in the images. Some agglomerates can be seen around one cell in Fig. 31.K.

The SiO2-NM200 agglomerates are found to a certain distance with the cells, leading to think that no physical attachment between both of them occurs. The analytical study of the material from the observable agglomerate in Fig. 31.O. confirms its silica composition.

Comparing the images of the NMs interaction between R. subcapitata and D.

subspicatus, there is a clear difference in the amount of NMs agglomerates around the algae cells. D. subspicatus showed lesser attachment of the tested NMs and lesser agglomerates are found around the cells for the same concentration. These results confirm the differences found in the flow cytometry analysis.

For many of the images of the algae-NMs interaction, the NMs agglomerates found around the algae kept a margin of distance to the cell wall. This could be due to an attachment not directly to the cell wall but to the exopolymeric substances (EPs) produced by the algae.

Evidence of this can be found in the work of [START_REF] Zhao | Interactions of CuO nanoparticles with the algae Chlorella pyrenoidosa: adhesion, uptake, and toxicity[END_REF] and [START_REF] Adeleye | Interactions between Algal Extracellular Polymeric Substances and Commercial TiO2 Nanoparticles in Aqueous Media[END_REF] showing that the interaction between EPS and TiO2 NMs is driven by electrostatic interactions and chemical bonding. This statement agrees with what was found in this chapter's article.

Algae toxicity test

The following toxicity data was generated by and belong to the NANoREG2 project.

The microalgae growth inhibition test was performed on Raphidocellis subcapitata according to the OECD 201test guideline (OECD, 2011b). The tested concentrations were 0, 0.1, 0.3, 1.0, 3.2, and 10 mg/L. The EC10, EC25 and EC50 values are presented in Table 8. The Log-normal model in the REGTOX software for Microsoft Excel (Vindimian, 2005) was used for the calculation of toxicity parameters (ECx). Since the toxicity data for all the same set of NMs of this work was determined for the NANoREG2 project, the aim was to analyze the possible correlation between the algae toxicity and the algae-NMs interactions. But no correlation could have been found. On the other hand, possible relations between the toxicity data and the NMs physicochemical characteristics were able to be identified. The TiO2 NMs that have rutile form (TiO2-NM103 and TiO2-NM104) are less toxic than the anatase formed NMs. TiO2-NM101 is the more toxic of the TiO2 NMs, this NM has also the smallest pristine size of the set. ZnO-NM110 showed the highest toxicity of the entire set, which can be probably caused by its dissolution and release of Zn ions. It has been frequently observed that zinc oxide NMs present high dissolution in the media (Bian et al., 2011[START_REF] Reed | Solubility of nano-zinc oxide in environmentally and biologically important matrices[END_REF].

Main conclusions

Answering to this chapter's presented questions, it can be concluded that:

• The short-time contact test and subsequent flow cytometry analysis has allowed to characterize the algae-NMs interaction. Dose-response profiles were obtained for the 3 different microalgae species and the set of 12 NMs. The developed method showed consistency throughout the many independent repetitions for all the tested algae and

NMs. These results were also confirmed by optical microscopic observations and TEM images.

• The analysis showed that the algae-NMs interaction is dependent of the algae species (D. subspicatus showed less interaction with NMs than the other two tested species) and the type of NMs (different profiles for the tested NMs).

• The differences between the NMs interaction have shown to be dependent on the NMs composition (SiO2 showed no modification of the algae granularity), on the zeta potential (more negatively charged NMs interact less with the algae), and on the crystalline phase (for TiO2 rutile NMs interacted more with the algae). The influence of other NMs characteristics, as hydrophobicity, remains to be elucidated.

• The algae-NMs interaction as analyzed for this study does not evolve significantly during the first 2 hours of contact. It seemed to be almost an instant reaction, answering to an electrostatic attraction/repulsion principle.

• These findings are relevant in the NMs risk assessment since the OECD 201 test guideline proposes the usage of R. subcapitata or D. subspicatus as valid species. Since the interaction with NMs between the two species is not the same for a given NMs concentration, one should be careful when conducting the analysis, at least to use constantly the same species.

According to these findings, a new question arises:

Are the differences in the interaction of algae-NMs similar to other species from a different food chain level?

Chapter 4: Uptake and elimination of nanomaterials by Daphnia magna

Introduction and context

In the precedent chapter the interaction between different NMs and primary producers (microalgae) was analyzed. Following up in the trophic chain, the interest of study the interaction with the next level type of organisms arises. Microcrustaceans are important primary consumers that feed on microalgae and they, in turn, are the basis of feeding for secondary consumers such as fish.

Daphnia magna is a widely used species in ecotoxicological studies, it is the model organisms for the OECD 202 acute immobilization and reproduction test (OECD, 2004). The importance of studying daphnids in the context of NMs uptake and elimination determination comes from their quality of being a filter feeding species. Some micrometric NMs agglomerates can easily be mistaken for food and daphnids will avidly ingest them (Seitz et al., 2015).

A previous study that worked with a NM, similar to one of the NMs from the selected set of this thesis, could successfully determine the uptake and elimination kinetics using 1 mg/L as exposure concentration [START_REF] Zhu | Toxicity and bioaccumulation of TiO2 nanoparticle aggregates in Daphnia magna[END_REF]. Based on this study, a series of uptake and elimination experiments were conceived under the same conditions for all the set of NMs and a kinetic model was applied. Modeling the NMs uptake in organisms is essential to reinforce experimental research, develop overarching theories, improve the fundamental understanding of NMs exposure and hazard, and thus enable risk assessment of NMs [START_REF] Baalousha | Modeling nanomaterial fate and uptake in the environment: current knowledge and future trends[END_REF].

Kinetic models are recommended to be used in time-dependent exposure conditions [START_REF] Ockleford | Scientific Opinion on the state of the art of Toxicokinetic/Toxicodynamic (TKTD) effect models for regulatory risk assessment of pesticides for aquatic organisms[END_REF], and their resulting uptake and efflux rate constants have been applied to NMs exposure cases where steady-states are not achieved [START_REF] Fan | High bioconcentration of titanium dioxide nanoparticles in Daphnia magna determined by kinetic approach[END_REF] The main questions raised when assessing this issue are as following:

• How is the uptake behavior of the different NMs in the microcrustacean Daphnia magna?

• How these daphnids eliminate the NMs once they are no longer exposed?

• Do these behaviors can be generalized and expressed as functions through modelling to obtain rates of uptake and elimination?

• Is it possible to regroup the NMs according to their uptake and elimination behavior?

• Is it possible to identify some NMs characteristics that are involved in the differences of uptake and elimination behavior?

• How the findings about the NMs uptake and elimination behaviors can be relevant for risk assessment purposes?

The scope of this chapter is summarized in the following schematic figure.

Dissolution of ZnO-NM110

The dissolution profile of the ZnO-NM110 was analyzed under the same exposure conditions as the uptake phase of daphnids. Three containers of ISO media with 1 mg/L of ZnO-NM1100 were sampled at 0, 2, 6 and 24 hours. The samples were acid-digested and analyzed by ICP-OES. The profile shows a high dissolution behavior (Fig 34), even at the starting point of measure at T0 there is already a 30% of Zn dissolved, arriving at 84% at 24h.

Binocular loupe imaging study

At the end of the uptake phase (24h of exposure), organisms were sampled to be observed under a binocular loupe. Photos were taken of the whole body at 20x amplification (Figure 35) and detailed photos of the head and the carapace were taken at 90x amplification (Figures 36 and37).

At the exposure concentration of 1mg/L no big agglomerates around the daphnids could have been noticed. The whole-body photos show for the TiO2 and the ZnO NMs that the digestive tubes contain NMs which has a different aspect from the photos of the control that showed greener and les compact content. The head photos show some NMs agglomerates around the rostrum of daphnids, especially for TiO2-NM105. NMs agglomerates attached to the carapace surface can also be noticed in the photos of TiO2-NM100, TiO2-NM101 and TiO2-NM102. For the rest of the NMs no agglomerates are detected and the surfaces of the heads and the carapaces between the control and the exposed organisms showed no differences.

Main conclusions

Answering to this chapter's presented questions, it can be concluded that:

• The NMs uptake behavior during a 24h exposure to 1mg/L of each of the 12 different NMs presented a rapid increase of the NMs concentration in the daphnids during the first hours, arriving for most of the NMs to a plateau at the end of the exposure. Two

NMs presented a different kind of behavior: SiO2-NM202 did not show any uptake at any point of the exposure and ZnO-NM110 showed a particular uptake profile possibly linked to the zinc dissolution during the experience.

• For the those NMs that presented uptake, the elimination behavior was determined by a fast lost during the first hours due to excretion. This phenomenon was also promoted by the presence of food during the elimination phase. Remaining metal concentrations up to 8% of the maximal uptake concentration can be found in the organisms after 5 days of elimination.

• A toxicokinetic model was applied and the uptake and elimination rates were determined. The model proved to be adequate to be applied to the studied kind of exposure and elimination experience, presenting validated goodness-of-a-fit criterion for all the NMs for which it was applied.

• Some NMs could have been clustered by their similar uptake or elimination rates.

Groups of low, intermediate and high kuw and kee were proposed.

• Some NMs physico-chemical characteristics can be linked to the uptake and elimination behaviors, as the dissolution of ZnO NMs that can explain the particular uptake profile presented by this NM.

• These results can be relevant in the NMs risk assessment since the similarities in the uptake rates of some NMs can be considered for grouping strategies.

At this point, and after the aforementioned conclusions about the uptake and elimination behavior of NMs in the cladoceran D. magna, a new question arises:

Which are the toxic effects related to that NMs-daphnid experience?

The scope of this chapter is summarized in the following schematic figure

Introduction

The fact that nanomaterials (NMs) have specific mechanical, catalytic and optical properties has caused an exponential development and growth of new manufactured or engineered NMs over the past few decades (Klaine et al., 2008). Metal oxide NMs specially exhibit fascinating electronic and magnetic properties, being among the most produced [START_REF] Hendren | Estimating Production Data for Five Engineered Nanomaterials As a Basis for Exposure Assessment[END_REF], and the most emitted NMs (Keller and Lazareva, 2014). Because of their widespread production, use, and emission; these NMs released in the environment represent a potential risk for living organisms in aquatic and terrestrial ecosystems [START_REF] Hyseni | Toxicological Effects of Nanomaterials on Aqueous and Terrestrial Ecosystems[END_REF]. The bioavailability and bioaccumulation of NMs depend on combined influences of NMs concentration, nature of the environment, route of exposure, biology and functional ecology of the organism [START_REF] Luoma | Why Is Metal Bioaccumulation So Variable? Biodynamics as a Unifying Concept[END_REF]. The major route of NMs uptake for aquatic organisms is through adsorption or ingestion [START_REF] Hou | Biological accumulation of engineered nanomaterials: a review of current knowledge[END_REF].

NMs toxic effects towards aquatic organisms are complex and can affect at different biological levels (physiological cellular, molecular and genomic), they can even affect the behavior (individual level) of some organisms (Boros and Ostafe, 2020). Additionally, NMs have already been proven to display specific nanoparticulate-related toxicity towards organisms differently as compared to their corresponding ions [START_REF] Genevière | Responses to iron oxide and zinc oxide nanoparticles in echinoderm embryos and microalgae: uptake, growth, morphology, and transcriptomic analysis[END_REF]. To study the effects of NMs at cellular and individual levels, many techniques have been developed as the use of cellular and molecular tools. Among them, the transcriptomic analysis contributes significantly in deciphering the molecular mechanisms against NMs stress, by establishing a connection between the expression of genes and cellular metabolism [START_REF] Singh | Abiotic stress tolerance in plants: growth regulators and transcriptional control of multiple signaling pathways[END_REF]. Recently, transcriptomic tools have been proposed as a suitable approach for investigating ecotoxicity of NMs as they allow to screen many endpoints in a relatively short amount of time [START_REF] Barrick | Investigating a transcriptomic approach on marine mussel hemocytes exposed to carbon nanofibers: An in vitro/in vivo comparison[END_REF]. This High Throughput Screening (HTS) method is becoming more and more relevant to identify the toxicity pathway of these contaminants in a regulatory perspective [START_REF] Châtel | Transcriptomic approach: A promising tool for rapid screening nanomaterial-mediated toxicity in the marine bivalve Mytilus edulis-Application to copper oxide nanoparticles[END_REF].

Once the NMs is inside the organisms and in contact with the cells, they are going to be stopped by the cell membrane that limits the entry of many materials, but there are some structures and mechanisms that allow the passage of materials including NMs (Châtel and Mouneyrac, 2017). One process that allows the translocation of intact NMs into the cell is endocytose [START_REF] Fabrega | Silver nanoparticles: Behaviour and effects in the aquatic environment[END_REF]. NMs may then, interact with intracellular components such as proteins, lipids or nucleic acids; inducing the production of reactive oxygen species (ROS) [START_REF] Cheng | Nano-bio effects: interaction of nanomaterials with cells[END_REF] which is the most studied nanotoxicity mechanism [START_REF] Vale | Manufactured nanoparticles in the aquatic environment-biochemical responses on freshwater organisms: A critical overview[END_REF].

Oxidative stress can damage cell functions including oxidative modification of proteins, lipid peroxidation, DNA-strand breaks, modification to nucleic acids, activation of redox-sensitive transcription factors, modulation of inflammatory responses through signal transduction, leading to cell death and genotoxic effects (Fu et al., 2014) [START_REF] Chiang | Nanoscale ZnO Induces Cytotoxicity and DNA Damage in Human Cell Lines and Rat Primary Neuronal Cells[END_REF] [START_REF] Poli | Oxidative Stress and Cell Signalling[END_REF]. NMs are also responsible to impact protein expression [START_REF] Gomes | Differential protein expression in mussels Mytilus galloprovincialis exposed to nano and ionic Ag[END_REF]. At an individual level, inhibition on the daphnids molting caused by TiO2 NMs has been reported; molting being an essential process for the development of species as microcrustaceans, molting disruptions can lead to high levels of immobility and ultimately to mortality (Dabrunz et al., 2011). Additionally, impairments in the reproduction of D. magna exposed to NMs have been reported (Hartmann et al., 2019) [START_REF] Judzińska | First step in reproduction disorder pathway assembly induced by nanomaterials in Daphnia magna[END_REF].

The cytotoxicity of the NMs is related to several factors, including material composition, size, and surface ligands [START_REF] Cheng | Nano-bio effects: interaction of nanomaterials with cells[END_REF]. Through the "omics" techniques, it has been possible to distinguish and differentiate the effects of exposures between different NMs compositions, sizes and surface coatings [START_REF] Poynton | Insights from 'Omics on the Exposure and Effects of Engineered Nanomaterials on Aquatic Organisms[END_REF]. But the attempt to relate the physicochemical properties of NMs to organisms' toxicity has been described as controversial because the correlation may not imply causation [START_REF] Skjolding | Aquatic Ecotoxicity Testing of Nanoparticles-The Quest To Disclose Nanoparticle Effects[END_REF]) (Vale et al., 2016) (von Moos andSlaveykova, 2014). That is why comparative studies of NMs with different physicochemical characteristics, but under the same conditions of organisms' exposure, are necessary to examine the possibility and the limits of a correlation and determine if some specific NMs properties can be linked to some specific toxic effects. Additionally, to our best knowledge, systematic studies that evaluate the cellular as well as the individual effects of NMs, during exposure and after elimination are scarce, and they are necessary for the global understanding of NMs-organisms interaction.

The principal objective of this systematic study is to determine and compare the effects of twelve (different metallic oxide) NMs on the gene expression of D. magna for a non-lethal 1-day exposure and after an elimination period. The set of targeted genes includes cellular and individual processes in which NMs are known to interact with. The second aim is to compare the mechanisms that can be disturbed between uptake and elimination phases. And a final goal is to study if certain transcriptomic alterations can be related to the amount of NMs accumulated by the organisms during uptake or remaining after elimination; or related to the NMs characteristics.

Materials and Methods

2.1.Test organism

For this study the freshwater microcrustacean Daphnia magna Straus was used. The INERIS facilities permanently breed a synchronous population of D.magna in an Elendt M4 media with daily feeding of freshwater microalgae (Chlorella vulgaris, Desmodesmus subspicatus and Raphidocellis subcapitata) at 2 mg/L TOC. The organisms were raised in 2 L aquariums at 20 ± 2°C with a day-night cycle of 16h of light (1000-1500 lux).

Nanomaterials preparation and characterization

The

Acute toxicity

The acute toxicity experiment was performed according to the OECD 202 test for acute immobilisation on D. magna (OECD, 2004). For all the tested NMs the concentrations were 0.0; 0.1; 0.3; 1.0; 3.2; 10.0; 31.6 and 100 mg/L in the ISO media. The EC10, EC25 and EC50 were calculated.

Exposure and elimination experiments

For each uptake and elimination experiment, daphnid neonates were put in 4L aquariums with M4 media. For one week the daphnids were fed with the freshwater microalgae Raphidocelis subcapitata at an equivalent concentration of 1 mg/L TOC. When the organisms arrived at their 8 th day, the uptake phase started. Prior to the exposure, the daphnids were feed one last time, after one hour they were placed in clean ISO media for acclimatization and to clean their guts, during one hour.

Organisms were placed by groups of 50 individuals in 1L containers in triplicates with ISO media spiked with 1mg/L of each tested NMs. Non-contaminated aquariums were used as controls for all NMs exposures. Organisms were exposed for 24 hours and samples were taken at the end of the uptake phase from the exposed and the controls aquariums. Then, the remaining daphnids were placed in clean ISO media for a 5 days elimination phase; they were fed daily with R. subcapitata 1.7 mg/L TOC, and samples of the tested and control aquariums were collected at the end of the elimination phase. All samples were immediately frozen at -80°C waiting for the transcriptomic analysis.

For all the tested NMs experiments, the number of dead organisms per aquarium were counted and samples were also taken for metal dosing at the end of the uptake phase and at the end of the elimination phase in independent triplicates. Acid digestion of samples was done using a microwave (Mars6, CEM) in Teflon reactors adding 3 mL of ultra-pure HNO3 and 2 mL of HBF4 for TiO2, ZnO and CeO2 samples or HF for SiO2 samples which was afterward neutralized with 20 mL of 55 g/L ultra-pure H3BO3. After digestion, all samples were adjusted to a final volume of 50mL and analyzed using an inductively coupled plasma optical emission spectrometry (ICP-OES; Agilent Technologies 5110).

2.5.RT-qPCR assay

RNA extraction and reverse transcription

Each sample consisted in a pool of 6 organisms. Concerning RNA purification, Trizol

Reagent ® (Ref: 15596026, Invitrogen ™) was added to each sample (1mL for 100 mg of sample), which was mechanically grinded until no remains could be seen. Then, the samples were centrifugated 10 minutes at 12000g at 4°C and the supernatants recovered. After 5 min at room temperature, chloroform was added (0,2mL/mL of Trizol) to each sample, then a centrifugation of 15 minutes at 12000g at 4°C allowed to recover the upper aqueous phase.

Isopropylic alcohol was then added (0,5mL/mL of Trizol) and after 10min at room temperature, the samples were centrifugated 10 minutes at 12000g at 4°C. The RNA pellet was washed with 200µL ethanol 95% then dried and finally diluted in 15 µL of RNase/DNase-free water. The RNA concentration of each sample was determined using a NANODROP (Thermo Scientific ™ NanoDrop 2000). The reverse transcription was realized with 1.3 μg of purified RNA using oligo-dT primers following the SuperScript ™ III First-Strand Synthesis SuperMix protocol supplied by Invitrogen™.

RT-qPCR analysis

The cDNA amplification was performed using a LightCycler 480 Real Time PCR system (Biorad) and the kit SYBR Green Power Master Mix (Invitrogen™). D. magna primers of 4 housekeeping genes and 11 target genes were chosen and their sequences were taken from published literature as detailed in Table 1. The choice of the target genes was based on trying to achieve a wide range of metabolic processes, covering cellular as well as individual levels.

Each sample was analyzed in triplicates. The setup program for the amplification had a first step of 10 minutes at 95°C for the enzyme activation, followed by 50 cycles of amplification/quantification (one cycle: 30s at 95°C, 30s at 58°C, 30s at 72°C). At the end of the cycles, the melting curves were analyzed starting at 60°C up to 95°C with readings every 0.5°C. Once all samples have been analyzed and their resulting Ct obtained, the gene amplification values were calculated based on the 2 (-𝛥𝛥𝐶𝑡) principle (Livak and Schmittgen, 2001) and the calculation used the geometric mean of multiple housekeeping genes following the GeNorm method (Vandesompele et al., 2002).

Statistics

Transcriptomics and mortality data were analyzed with RStudio (version 1.3.1073). For the transcriptomics data, preliminary tests of normality (Shapiro-Wilk's test) and variance homogeneity (Levene Test) were carried out. Then, according to the results of those tests, the Kruskal-Wallis test for non-parametrical data was applied (significance of p < 0.05). For the analysis of the mortality data, a generalized linear model for binomial data was used; the pvalues of the z-test of comparison to the control were corrected with a Bonferroni correction within each dataset. For the acute toxicity data, the Log-normal model in the REGTOX software for Microsoft Excel (Vindimian, 2005) was used for the calculation of toxicity parameters (ECx) and their confidence intervals. ECx values are reported with 95% confidence intervals.

Results

NMs characterization

All the 12 tested NMs were characterized by DLS for the hydrodynamic diameter and Zeta potential in the ISO media. General physico-chemical properties are already given by JRC (Manier et al., 2013). Table 2 resumes the main characteristics of these NMs. Pristine sizes vary between 6 nm for TiO2-NM101 and 151 nm for ZnO-NM110. Specific surface area varies from 9.23 m 2 /g for TiO2-NM100 to 316.07 for TiO2-NM101. Different crystalline phases are present among the tested NMs: anatase and rutile for TiO2, hexagonal for ZnO, amorphous for SiO2 and cubic for CeO2. Three of the tested NMs were coated. Hydrodynamic diameter measures in ISO media (pH 7.8 ± 0.2) showed that the NMs suspensions were formed for more or less bigger agglomerates starting at 361 nm and arriving at around 2µm. The Z potential of the suspensions presented mostly negatively charged NMs with TiO2-NM103 and TiO2-NM104 being the only two positively charged (3.9 and 4.7

mV respectively), and the silica NMs the most negatively charged.

3.2.Acute toxicity

Acute toxicity results of the OECD 202 test of D. magna 48h exposure to NMs are summarized in Table 3. Almost all the tested NMs showed values of EC10, EC25 and EC50 above 100 mg/L; only TiO2-NM101 (14.8; 24.1 and 39.1 mg/L respectively) and ZnO-NM110 (9.43; 14.58 and 22.48 mg/L respectively) caused significant inhibition of the organism mobility in the range of the tested concentrations for the OECD 202 acute toxicity test.

Mortality and metal dosing

After 24 hours of exposure to NMs, the mortality registered by the groups exposed to the NMs was not different from the control groups, showing that this study is conducted at a sublethal concentration exposure. Moreover, after 5 days of elimination, only the SiO2-NM204 group has led to a significant mortality (p = 2.6e-11) as shown on Figure 1.

The samples at the end of the uptake and elimination phases were analyzed by ICP-OES and the body burden in g/Kg of dry weight were found as showed on Table 4. For the uptake phase, all NMs showed an uptake except for SiO2-NM202 (confirmed by 3 independent replicates). ZnO NMs exhibit a low uptake with a concentration in the sample of 0.67 g/Kg.

TiO2-NM103 presented the highest uptake level with 114.23 g/Kg. For the elimination phase, it can be noticed that after 5 days of elimination a residual concentration is still detectable inside the organisms, the highest values corresponding to TiO2-NM105 and SiO2-NM204 (1.82 and 2.01 g/Kg respectively). Organisms exposed to SiO2-NM200 seemed to have eliminated all the ingested NMs, since no residual NMs were detected (< limit of quantification) for this group.

RT-qPCR analysis

The gene expression was calculated for all the 12 tested NMs corresponding to the exposure and elimination phases. The targeted genes include metabolic processes at cellular and individual level as shown on Figure 2. At the cellular level, the studied genes are involved in oxidative stress and detoxication (sod, gst, cyp, hsp70, mt), DNA repair (rev1) and endocytose (flot). At the individual level, the studied genes include processes like developing and molting (jhe, ecr), reproduction (vtg) and digestion (amy). Four housekeeping genes (act, 18s, gpdh and ubc) were analyzed for all the samples, but the expression of act was not stable all along the samples (GeNorm method), therefore it was not considered for normalization. Thus, the relative quantification of gene expression was normalized using 3 housekeeping genes, for a more robust analysis. During the uptake phase, the differential gene expressions (DGE) showed a downregulation tendency for TiO2-NM100, TiO2-NM101, TiO2-NM102, ZnO-NM110, SiO2-NM204 and CeO2<10nm. Upregulation was shown for most genes analyzed in organisms exposed to TiO2-NM103, TiO2-NM104, TiO2-NM105, SiO2-NM200 and SiO2-NM202.

CeO2<25nm showed very little DGE with only cyp, flot and ecr genes expressions affected.

Regarding the magnitude of the DGE in the uptake phase, TiO2-NM101 has the most extreme folding differences compared to the other tested NMs, with a high upregulated mt expression and highly downregulated expressions for sod, rev1, flot, and for all the genes concerning individual processes. It is important to note that this NMs has the higher acute toxicity values of the TiO2 NMs (Table 3). High DGE values are also present for ZnO-NM110 that has also shown greater acute toxicity than the other NMs. High upregulation is also present in TiO2-NM105 vtg, SiO2-NM202 sod, and SiO2-NM200 mt, jhe and amy gene expressions.

For the TiO2 NMs, two types of behavior in the DGE can be noticed during the uptake phase: mostly downregulation of genes for organisms exposed to TiO2-NM100, TiO2-NM101 and TiO2-NM102 (all three of them have anatase crystalline form) and mostly upregulation for TiO2-NM103, TiO2-NM104 and TiO2-NM105 (that have rutile crystalline form for TiO2-NM103 and TiO2-NM104 and a mix of rutile and anatase for TiO2-NM105).

Regarding the elimination phase, less genes presented DGE compared to the uptake phase and the main tendency is mostly to upregulation. Nevertheless, some NMs gene expressions showed downregulation, especially for ZnO-NM110, SiO2-NM202 and SiO2-NM204. Two NMs showed very little DGE arriving at this phase: SiO2-NM200 that had only DGE for hsp70, rev1 and flot and CeO2<25nm for sod, cyp and mt.

Regarding the DGE magnitude in this elimination phase, the highly upregulated genes were ZnO-NM110 and CeO2<25nm mt, CeO2<10nm sod and vtg. And the highly downregulated gene expressions were TiO2-NM105 sod and vtg. Concerning the gene expression across all the NMs, the genes that present the most affected folding expressions at the end of the elimination phase are sod, mt and vtg; meaning that the processes still affected at this point are mainly oxidative stress, metal binding and reproduction.

The different behaviors of the anatase and rutile NMs in the DGE expressions can still be found during the elimination phase. The anatase NMs still have some downregulated gene expressions and the rutile NMs only induced upregulated mRNA levels. But TiO2-NM105 has a different behavior, presenting more downregulated genes and being more similar to the behavior of the anatase-formed NMs.

No correlation could have been found between the body burden in the organisms at 24h and the DGE at the uptake phase, nor between the body burden at 120h of elimination and the DGE for the elimination phase (statistical analysis in supplementary data).

Discussion

This systematic study aimed to determine and compare the exposure general effects and specially the transcriptomic differential gene expressions of the microcrustacean D. magna to a set of 12 different metallic oxide NMs. The expression of several genes involved in essential functions for the organisms were studied through RT-qPCR, allowing to produce a large quantity of data in a time and cost-effective manner. In particular, the expression of genes involved in oxidative stress, detoxication, DNA repair, endocytose, development, molting, reproduction and digestion were evaluated (as shown in Figure 3), after 1-day exposure to NMs and after 5 days of elimination. Changes in the expression of genes involved in these different processes at the cellular and individual levels present an overview of the potential consequences for the organism.

Actin, one of the 4 housekeeping genes chosen for this study, showed to be not stable across the analysis. This can be an evidence of cytoskeleton filament disruption by the NMs.

Other studies have shown that in vitro exposures can affect the cell permeability by disruption of the actin cytoskeleton for silica NMs (30nm) [START_REF] Cornu | Small silica nanoparticles transiently modulate the intestinal permeability by actin cytoskeleton disruption in both Caco-2 and Caco-2/HT29-MTX models[END_REF], for ZnO NMs [START_REF] Paszek | Zinc Oxide Nanoparticles Impair the Integrity of Human Umbilical Vein Endothelial Cell Monolayer In Vitro[END_REF] and for TiO2 NMs that have even shown actin-mediated internalization into the cells (Huerta-García et al., 2019).

The results of the present study showed a general toxicity response for all the NMs, especially during uptake where most of the process were affected without specific targets, the most toxic response was for TiO2-NM101 that showed cellular distress and general alteration of all the gene expressions. For the elimination phase, the gene expression results presented lower toxicity responses and more specific affected targets. Several genes were downregulated during the uptake phase and most genes are upregulated during the elimination phase. In contrast, [START_REF] Arini | Bioaccumulation dynamics and gene regulation in a freshwater bivalve after aqueous and dietary exposures to gold nanoparticles and ionic gold[END_REF] had the opposite behavior when exposing clams to Au NMs and studying some similar cellular targets (oxidative stress, endocytosis, metal biding), being most genes upregulated during uptake and downregulated during the elimination.

Figure 3. Illustration of the simplified pathways in which the proteins corresponding to the targeted genes are involved and their interaction with NMs. The NMs enter the cell by endocytose that can be mediated by flotillin (flot). Once in the cell, the NMs can set off the production of reactive oxygen species that will be neutralized by the superoxide dismutase (sod), also activating detoxication mechanisms involving for example the cytochrome P450 (cyp) and the glutathione S transferase (gst). The presence of metal will be taking in charge by the metallothionein (mt). The potential genome damages will be repaired by the DNA repair gene (rev1) encoded protein and the heat shock protein (hsp70) will preserve the correct protein folding. At an individual level, genes involved in the developing and molting are studied encoding the juvenile hormone esterase (jhe) and the ecdysone receptor A (ecr). Vitellogenin (vtg) will inform about the reproduction effects and amylase (amy) about the digestive activity.

During the uptake phase a marked difference could be noticed between the DGE of TiO2-NM100, TiO2-NM101, TiO2-NM102 (anatase form) and TiO2-NM103, TiO2-NM104, TiO2-NM105 (rutile or mix rutile-anatase). This can be explained by the fact that, it has been proved that anatase forms are more toxic to D. magna than rutile forms (Clément et al., 2013).

Additionally, the photocatalytic activity of TiO2 NMs, when exposed to light radiation, has exhibited generation of radical species on daphnids [START_REF] Dalai | Acute Toxicity of TiO2 Nanoparticles to Ceriodaphnia dubia under Visible Light and Dark Conditions in a Freshwater System[END_REF] and it has been showed that anatase forms have more photocatalytic activity than rutile forms [START_REF] Luttrell | Why is anatase a better photocatalyst than rutile? -Model studies on epitaxial TiO2 films[END_REF].

Additionally, it is important to notice that both rutile forms are also coated, which may prevent the interaction with the organism reducing their toxicity, as explained by [START_REF] Gatoo | Physicochemical Properties of Nanomaterials: Implication in Associated Toxic Manifestations[END_REF].

In this study, TiO2-NM101 exposure led to the highest DGE for almost all genes during the uptake phase, this response profile can be correlated to the results of the acute toxicity test for this NM, since its values of ECx showed that this NMs is the most toxic among the TiO2 NMs and the second most toxic among the studied NMs. A specificity of TiO2-NM101 is that it has the larger specific surface area among the set of NMs, and this may be related to its higher toxicity since TiO2 particles with larger surface area can generate more hydroxyl radicals; and [START_REF] Xiong | Specific surface area of titanium dioxide (TiO2) particles influences cyto-and phototoxicity[END_REF] found that higher cytotoxic effects have been displayed for TiO2 NMs with larger specific surface areas.

Concerning the oxidative stress process, daphnid exposure to TiO2 NMs caused upregulation of genes associated with antioxidant activities, such as superoxide dismutase (sod), glutathione peroxidase (gpx), catalase (cat) [START_REF] Wong | A Comprehensive Review on the Aquatic Toxicity of Engineered Nanomaterials[END_REF]. In the present study DGE on the expression of the rev1 gene for all the tested TiO2 NMs are present. DNA damage caused by TiO2 has been reported by various authors [START_REF] Girardello | Titanium dioxide nanoparticles induce genotoxicity but not mutagenicity in golden mussel Limnoperna fortunei[END_REF] [START_REF] Shi | Immunotoxicity of nanoparticle nTiO2 to a commercial marine bivalve species, Tegillarca granosa[END_REF].

Similarly to our results, in a study of exposure of D. magna to 0.1 mg/L ZnO NMs for 14 days, affectations in the regulation of genes as sod, gst and vtg are shown and specially upregulation of gst gene at 1-day exposure is presented [START_REF] Chen | Comparison on the effects of water-borne and dietary-borne accumulated ZnO nanoparticles on Daphnia magna[END_REF]. ZnO NMs are known to dissolve and release Zn ions (Bian et al., 2011) and some of the toxic effects typically found on metal ion cytotoxicity can be found after ZnO NMs exposure. Besides, Zn is a trace element naturally found in the organisms and it has been found that they can regulate their Zn internal concentration [START_REF] Adam | The uptake and elimination of ZnO and CuO nanoparticles in Daphnia magna under chronic exposure scenarios[END_REF]. This can explain the low uptake of ZnO NMs found in the daphnids, since this organism is able to detoxicate and eliminate the Zn from ZnO NMs faster than any other tested NMs. It can also explain the lack of difference in the expression of the metallothionein gene during the exposure phase, although a strong upregulation is exhibited during the elimination phase which may correspond to the Zn detoxication by the organisms.

Concerning the development process, DGE is present for the jhe and ecr genes suggesting that the organism's growth can be affected. Specially, jhe expression is disrupted for all the tested TiO2 NMs in the uptake phase. TiO2 NMs are reported to cause delay in molting of daphnids inducing a negative effect in development (Dabrunz et al., 2011). Additionally, exposure to CeO2 NMs have shown to affect the growth of D. magna mainly due to increased energy consumption for feeding and depuration of non-nutritious NMs [START_REF] Gaiser | Interspecies comparisons on the uptake and toxicity of silver and cerium dioxide nanoparticles[END_REF]. In our study, organisms exposed to CeO2<10nm showed more DGE for jhe and ecr than those exposed to CeO2<25nm.

In regard to the digestive process, the results for amylase gene showed DGE specially during the exposure phase and particularly for daphnids exposed to TiO2 NMs after the elimination phase. It has been shown that in D. magna, TiO2 NMs caused disruption of digestive enzymes, such as amylase and esterase, affecting nutrient assimilation and energy allocation (Boros and Ostafe, 2020). Additionally, NMs of a certain size (240 -640 nm) can be mistaken for food by daphnids (Seitz et al., 2015), and this can result in an increased filtering activity [START_REF] Rehse | Short-term exposure with high concentrations of pristine microplastic particles leads to immobilisation of Daphnia magna[END_REF]; thus, the digestive activity can be impacted. Surprisingly, one of the tested SiO2 NMs (NM202) did not show any uptake, nonetheless the gene regulation effects for this NMs are very important; after the uptake phase most of the genes showed DGE, but especially after the elimination phase all the gene expression involved in the studied cellular and individual processes are affected with the only exception of flot.

Deepen studies will be needed to identify the peculiarities of this NMs-organism interaction that resulted in this unexpected outcome. Additionally, the higher mortality of daphnid that occurred for the SiO2-NM204 experimental groups after the elimination phase can be related to the results of high downregulation of gene expression at cellular level differencing this NMs related effects from the other tested silica NMs of this study.

For further studies, a register of the daphnid's molts as well as individual's size register could be used to confirm the observed effects on the gene regulation corresponding to the development processes. Finally, comparative multigenerational studies on sublethal concentration exposures may be useful in establishing the long-term effects and transcriptomic disturbances of NMs; some studies have dabbled in this area such as the one of [START_REF] Maselli | Multigenerational effects and DNA alterations of QDs-Indolicidin on Daphnia magna[END_REF] with QDs, the one of (Hartmann et al., 2019) with Ag and TiO2 NMs or the one of (Ellis et al., 2021) who found no transgenerational effects after the exposure of D. magna to the TiO2-NM105.

Conclusion

This work presents a systematic study of a set of 12 different NMs under the same experimental conditions and the obtained results constitute a rich database that informs about important processes in which gene expression is affected during the uptake and elimination of NMs and that can be used for regulatory purposes. This transcriptomic analysis has provided informative and comparative knowledge about the NMs-induced toxicity at gene expression level for a sublethal concentration exposure. And it can be concluded that, all the studied cellular and individual processes were transcriptionally affected at some point by the exposure to every one of the 12 tested NMs and most rested disturbed after 5 days of elimination process.

During the uptake phase, downregulation of gene expressions was more common, and after 5 days of elimination it was upregulation.

During the uptake phase, TiO2-NM101 showed the highest DGE and more acute toxicity than most of the tested NMs. Additionally, during this phase two behaviors among the TiO2 NMs could be identified, mainly downregulation for the anatase formed NMs and upregulation for the rutile and mix of rutile and anatase formed NMs. SiO2-NM200 and CeO2<25nm can be identified as the ones that had lesser disruptions remaining after elimination.

Evidence of oxidative stress defense, repair of damaged DNA, restoration of misfolded protein, metal detoxication and endocytosis affectation were identified through gene expression variation at the cellular level for the effects caused by NMs. At the individual level, effects on the developing, molting, reproduction and digestion were transcriptionally evidenced specially at the end of the elimination phase.

No correlation between NMs uptake and gene expression modulation was found and SiO2-NM202 that did not show any NMs uptake, was the one that presented more effects between the tested silica NMs. The results on the effects of the gene expression modulation have shown many variations that may seem to have a connection with different NMs characteristics as composition, crystalline form (for TiO2) or even coating. Confirmation and detailed understanding of these phenomena are still needed Acknowledgements: The authors thank the support of the French Ministry for the Ecological and Inclusive Transition. A special recognition is given to Mélanie BRUNEAU for the assistance with the transcriptomic analysis.

Main conclusions

Answering to this chapter's presented questions, it can be concluded that:

• During the exposure phase, both level of biological organization (cellular and individual) are impacted, and downregulation of genetical expressions is more common during this phase. Evidence of oxidative stress defense, repair of damaged DNA, restoration of misfolded protein, metal detoxication and endocytosis affectation were identified as impacted processes at the cellular level; and at an individual level alterations in the genetic expression of genes related to developing, molting, reproduction and digestion were also determined. One NM showed extremely high gene expressions deregulations, the TiO2-NM101.

• During the elimination phase, the differential genetic expression was less important, and the effects turned to mostly presented gene upregulations, showing that the organisms present some resilience to the impacts of NMs exposure. Nevertheless, some NMs still presented some high gene expression alterations for the processes of oxidative stress and metal binding. At an individual level, reproduction was the most affected process displaying important alterations for the vitellogenin gene.

• No correlation between the uptake and elimination behaviors and the transcriptomic results could have been stablished. Nonetheless, the high deregulation on gene expressions of TiO2-NM101 can be connected to the fact that, this NM presented more toxic values of EC50 for the OECD202 test than almost all the other NMs studied. Additionally, the high mortality that occurred during the elimination phase of the experiment for SiO2-NM204 can be linked to some highly downregulated gene expressions that this group exhibited during this phase.

• Some NMs characteristics seems to be related to the differential genetic expressions presented by the organisms, in particular the crystalline form and coating, since both rutile formed and coated TiO2 NMs showed different responses from the rest of the TiO2 NMs.

• The obtained data can help to fill the gaps to assess the regulatory use of transcriptomics as a relevant method for early determination of NMs toxic effects and screening of NMs for grouping purposes.

After the interaction between NMs and algae and microcrustaceans has been studied and their effects described; there were all along some inklings that that a connection with the NMs physico-chemical characteristics could explain some features of these nano-bio interactions.

Then, the question that follows is:

Is there a correlation between some NMs physico-chemical characteristics and the obtained nano-bio behaviors that may allow to regroup the selected NMs?

The scope of this chapter is summarized in the following schematic figure.

Nanomaterials characteristics, nano-bio interactions and early molecular effects as elements for grouping strategies (in prep.)

Andrea RIVERO ARZE, Catherine MOUNEYRAC, Amélie CHATEL, Nicolas MANIER

Abstract

Since there is a great diversity of nanomaterials (NMs) already produced and expected to be produced, thanks to their numerous applications, the risk assessment of these materials has become an enormous task if wanted to be addressed in a case-by-case basis. Thus, the need of grouping and read-across approaches has become evident. Governments and regulatory bodies had started many projects focusing on finding grouping strategies for NMs. And, while some progress has been made, there are still many gaps and questions about the parameters that need to be considered for a good NMs grouping determination; especially for the parameters related to the interaction and effects of NMs on the biota. This work takes the principles of grouping approaches already developed by European Union projects as NANoREG2, and a large dataset of information about 12 different NMs, to analyze the grouping categories that can be made using the NMs physicochemical characteristics (intrinsic and media-dependent) and the nano-bio interaction information generated by the authors in previous works (34 parameters across 2 trophic level organisms). The results showed some NMs that were recurrently grouped together through the different analysis that were made, some NMs with similar physicochemical properties interact in a similar way with the organisms and have similar toxicity towards them.

This study highlights the importance of considering the nano-bio interactions for risk assessment and grouping purposes.

Keywords: Nanomaterials, grouping, ecotoxicity, microalgae, microcrustaceans.

Introduction

The diversity of engineered nanomaterials (NMs) keeps on growing thanks to their wide applications. There are NMs of different compositions, but within a composition category, there are NMs with multiple different characteristics as size, shape, surface chemistry, etc., and this has raised concerns about their risk assessment and regulation (Dekkers et al., 2016). Under the Registration, Evaluation and Authorization of Chemicals (REACH) regulation, in the European Union it is necessary to register and to provide a detailed report of risks for every substance manufactured at 1 ton per year or more (https://echa.europa.eu/regulations/nanomaterials).

Grouping and read-across approaches are already established for chemical substances to meet regulatory data requirements by deviation from performing standard test requirements for years (ECHA, 2008) (OECD, 2007). This approach is used to predict the outcome of an individual or a number of endpoints provided that appropriate data are available. But for NMs further aspects need to be considered since approaches based on molecular structural similarity alone are not sufficient to develop and verify grouping/read-across hypothesis that allow an adequate hazard assessment of NMs (Schwirn and Völker, 2019).

The case-by-case study of all the different existing and new NMs would require a lot of time and resources; and that is why, scientists, industrials and regulatory bodies are trying to find ways of grouping and read-across NMs with the aim of finding some properties that could provide reasonable prediction of NMs hazard without additional testing (Hund-Rinke et al., 2018). Based on this need for methodologies to group NMs, several initiatives have come to light in the last years. At the European Union level, research projects like, NANoREG (Gottardo et al., 2017), MARINA [START_REF] Bos | The MARINA Risk Assessment Strategy: A Flexible Strategy for Efficient Information Collection and Risk Assessment of Nanomaterials[END_REF], NanoTEST [START_REF] Dusinska | Towards an alternative testing strategy for nanomaterials used in nanomedicine: Lessons from NanoTEST[END_REF], Nanogenotox [START_REF] De | NANOGENOTOX, deliverable 7: identification of target organs and biodistribution including ADME parameters[END_REF], NANoREG2 (Mech et al., 2019) and GRACIOUS (Stone et al., 2020) were founded. And the European Chemicals Agency (ECHA) published a guidance document on the grouping of NMs (ECHA, 2019). Also, some governmental projects as nanoGRAVUR [START_REF] Wohlleben | The nanoGRAVUR framework to group (nano)materials for their occupational, consumer, environmental risks based on a harmonized set of material properties, applied to 34 case studies[END_REF], industrial [START_REF] Arts | A decision-making framework for the grouping and testing of nanomaterials (DF4nanoGrouping)[END_REF] and individual initiatives [START_REF] Gebel | Manufactured nanomaterials: categorization and approaches to hazard assessment[END_REF] [START_REF] Walser | Sameness: The regulatory crux with nanomaterial identity and grouping schemes for hazard assessment[END_REF] have proposed grouping strategies and case studies (ECHA/JRC/RIVM, 2016). Some grouping approaches tried to establish correlations between the intrinsic properties of NMs with some extrinsic properties (that depends of the surroundings), but the interdependence and correlations of the NMs properties could not be correctly established [START_REF] Arts | A decision-making framework for the grouping and testing of nanomaterials (DF4nanoGrouping)[END_REF]. Nevertheless, the grouping of NMs cannot rely solely on the intrinsic NMs properties; other extrinsic aspects as biological interactions, biokinetics, uptake and distribution, early and apical biological effects and life cycle aspects could be important but remain to be validated (Mech et al., 2019). Another challenge, that the NMs grouping attempts have faced, is the limited inter-comparability of data to work with [START_REF] Robinson | How should the completeness and quality of curated nanomaterial data be evaluated?[END_REF].

The approach developed by the NANoREG2 project consists in a grouping scheme that includes 3 pillar questions (Jantunen et al., 2018). The first is: What they are? That relates to the physicochemical (PC) properties of the pristine materials. The second: Where they go? Is about the system-dependent fate of the NMs, expressed as the PC characterization measures of the NMs in the media and/or the environment. And the third is: What they do? Which relays on the NMs hazards expressed as physical hazards, human toxicity and ecotoxicity data. This approach tries to establish correlations and linkages between the three pillars.

In what it concerns the NMs hazards data for the upper mentioned approach, the present study will focus on ecotoxicity data, and the analysis is centered on four classes of data that connect NMs and their interactions with the biota (see previous works (Rivero Arze et al., 2020), (Rivero Arze et al., 2021a) and [START_REF] Rivero Arze | Effects on Daphnia magna gene expression during exposure and elimination of twelve metallic oxide nanomaterials[END_REF])):

i)

The NMs-algae attachment interactions; and works from other authors showed that the attachment parameters between algae and NMs in short-term tests can be considered as a valuable tool for the identification of sets of similar nanoforms as requested by the ECHA in the context of grouping and read-across (Hund-Rinke et al., 2020).

ii) Uptake and elimination kinetics of NMs; and it has been pointed out that toxicokinetic information provides valuable mechanistic insights, and thus can be used to support grouping approaches (Mech et al., 2019) iii) Transcriptomic determination of early effects of NMs exposure in organisms; and the ECHA held a Topical Scientific Workshop on New Approach Methodologies in Regulatory Science which includes for example, high-throughput screening and omics, that may be used to confirm the mechanistic hypotheses for read-across (ECHA, 2016) iv) Ecotoxicity results from the OECD tests guidelines; for which it has been concluded that they can be applied to NMs by the OECD-WPMN (Working Party on Manufactured Nanomaterials) (OECD, 2009), although some adaptations were found to be necessary (Hund-Rinke et al., 2016), and that constitute one of the biggest source of ecotoxicity input for the existing NMs grouping approaches.

Based on the grouping concepts previously presented and the relevance of the broad dataset generated of nano-bio interaction in the grouping context, this work aims to establish groups of NMs based on potential explanatory links between the PC properties of NMs and their interactions with the studied organisms. At first, it is intended to group the NMs according to their PC pristine properties and PC measures in the tested media as a link between the first two pillars of the NANoREG2 approach. Then, the idea is to combine all the PC data with the nano-bio interaction and toxicity data (linking the first two components with the third component of the approach) to find out how these interactions with the biota intervene in the grouping results of the studied NMs.

Materials and Methods

All the data used for the analysis of this paper was generated in previous works, for detailed protocols refer to (Rivero Arze et al., 2020), (Rivero Arze et al., 2021a) and [START_REF] Rivero Arze | Effects on Daphnia magna gene expression during exposure and elimination of twelve metallic oxide nanomaterials[END_REF].The short version of the protocols is described below and the names of the chosen parameters for the present analysis are indicated in brackets and in italic.

Nanomaterials characterization

The set of studied NMs have been chosen came from the JRC Nanomaterial Repository and are considered as reference nanomaterials as they are well known and characterized. The The known characteristics of these NMs as pristine size (Prist.size), specific surface area (SSA), dissolution (Dissol) and reactive oxygen species production (ROS) were used in this document analysis. The NMs physicochemical (PC) characteristics of their fate in the different experimental media were also evaluated. Specifically; the hydrodynamic diameter in ultrapure water, algae media and daphnids ISO media (Size.stock, Size.algae and Size.ISO respectively), along with the surface charge (Zpot.stock, Zpot.algae and Zpot.ISO).

Test organisms

Three freshwater microalgae species, coming from the Culture Collection of Algae and Protozoa (CCAP), were used: Raphidocelis subcapitata strain CCAP278/4, Desmodesmus subspicatus strain CCAP276/22, and Chlorella vulgaris strain CCAP211/11B. They were cultivated following the OECD 201 test guideline recommendations (OECD, 2011).

The microcrustacean used for the experiments was the freshwater cladoceran Daphnia magna Straus. The organisms were raised in the laboratory facilities in M4 media and feed daily with the algae species previously mentioned at 2 mg/L TOC.

Algae-NMs interaction

Each of the three algae was put in contact with each NMs suspension during 15 min in a glass vial with constant stirring as described by [START_REF] Manier | Effect of two TiO2 nanoparticles on the growth of unicellular green algae using the OECD 201 test guideline: influence of the exposure system[END_REF]. The contact concentration was stablished at 5x10 5 cells/mL. The dose-response curves were obtained using 10 different concentrations as follows, 0.1; 0.5; 1; 2.5; 5; 7.5; 10; 12.5; 25 and 50 mg/L. All

Daphnids uptake and elimination kinetics and effects

For this experiment a synchronic population of daphnids that were 8 days old were exposed during 24 hours to 1 mg/L of the set of 12 NMs and then, they went through a phase of elimination of 5 days. Samples were collected at 0, 1, 2, 4, 6, and 24 hours of exposure and at 2, 6, 24, 48 and 120 hours of elimination. The samples were dried, weighted, acid digested and dosed using an inductively coupled plasma optical emission spectrometry (ICP-OES;

Agilent Technologies 5110). The uptake and elimination concentrations in the organisms were used to feed the MOSAICbioacc model (available at http://lbbe-shiny.univ-lyon1.fr/mosaicbioacc/), and the uptake and elimination rates were obtained (kuw; kee) as well as a kinetical bioconcentration factor BCFk. The data took from this part for the present analysis correspond to the maximal body burden at 24h of exposure (up24h), the remaining concentration after elimination (elim120h), and the modeled kinetic parameters (kuw, kee and BCF).

Samples were also collected to perform transcriptomic analysis at the end of the exposure phase and at the end of the elimination phase. The RNA of six organisms per sample was extracted using Trizol Reagent ® , and the reverse transcription was realized with 1.3 μg of purified RNA using oligo-dT primers following the SuperScript ™ III First-Strand Synthesis SuperMix protocol supplied by Invitrogen™. The cDNA amplification was performed using a LightCycler 480 Real Time PCR system (Biorad) and the kit SYBR Green Power Master Mix (Invitrogen™). Once all samples have been analyzed and their resulting Ct obtained, the gene amplification values were calculated based on the 2 (-𝛥𝛥𝐶𝑡) principle (Livak and Schmittgen, 2001) and the calculation used the geometric mean of multiple housekeeping genes following the GeNorm method (Vandesompele et al., 2002). The choice of the target genes was based on trying to achieve a wide range of metabolic processes, covering cellular as well as individual levels. At cellular level, the following genes were studied: endocytosis through the analysis of flotillin (flot), oxidative stress and detoxification processes with the genes of the superoxide dismutase (sod), the cytochrome P450 (cyp), the glutathione S transferase (gst) and metal binding with metallothionein (mt); DNA repair gene (rev1) and the heat shock protein (hsp70).

At the individual level the studied genes included: the juvenile hormone esterase (jhe), the ecdysone receptor A (ecr), vitellogenin (vtg) and amylase (amy).

Acute toxicity

The microalgae growth inhibition test was performed on Raphidocellis subcapitata according to the OECD 201test guideline (OECD, 2011). The tested concentrations were 0, 0.1, 0.3, 1.0, 3.2, and 10 mg/L. The EC50 values were calculated and used in this analysis as (OECD201).

The acute toxicity experiment in daphnids was performed according to the OECD 202 test for acute immobilization on D. magna (OECD, 2004). The concentrations of all the tested NMs were 0.0; 0.1; 0.3; 1.0; 3.2; 10.0; 31.6 and 100 mg/L in the ISO media. The EC10, EC25

and EC50 were calculated. The parameter used for this analysis is the EC50 (OECD202).

Statistical analysis

The statistical analysis in which this work is based is the Principal Components Analysis (PCA) carried out in RStudio (version 1.3.1073). The PCAs were performed on a matrix containing all the known and previously obtained parameters for all the 12 NMs. The complete matrix is presented in Supplementary Data. Some parameters were log-transformed : Prist.size, SSA, ROS, Dissol, Size.stock, Size.algae, Size.ISO, OECD201, OECD202, BCF, kuw, kee, and all the transcriptomic results.

Results and discussion

NMs similarities based on the physicochemical characteristics

The first analysis, following the NANoREG2 concepts, tries to understand the link that connects "what the NMs are" with "where they go". For this purpose, the physicochemical characteristics of the pristine NMs were analyzed with the measures of hydrodynamic diameter and surface charge in the different biological media used for the nano-bio interactions experiments. The results of the analysis are shown in Figure 1. In this PCA, the first axis is represented mainly by the parameters that describe the behavior of NMs in the media, as the surface charge in the ISO media, which is close correlated with the surface charge of the stock solutions and the hydrodynamic diameter in the algae media. The parameters that describe the nature of the NMs that are well represented in this analysis are the pristine size, logically anticorrelated with the specific surface area.

The resulting graphic of individuals allows to group the NMs in three clusters and some alone NMs that are different from the rest. The first cluster is formed by all the SiO2 NMs and the CeO2<10nm. This group is characterized by more negative surface charges and small hydrodynamic diameters. The second identified cluster is formed by both rutile and coated TiO2

NMs, NM103 and NM104 along with CeO2<25nm; which contrary to the first group are characterized by more positive surface charges and bigger agglomerates in the media. A third cluster formed by TiO2-NM102 and TiO2-NM105 can be identified. The most different NMs from the rest is TiO2-NM101specially characterized by its small pristine size and high SSA.

Opposite to this one is the NM110 with bigger pristine size.

NMs similarities combining NMs PC parameters and the biological interactions

In this part, both NMs PC characteristics (pristine and media-dependents) are analyzed along with the different nano-bio interaction and toxicity results obtained, in order to see if the are some correlations that can be determined and to investigate how the groups are formed in comparison with the previous results.

NMs-algae

The data from the NMs PC characteristics and the interaction results with microalgae as well as the ecotoxicological data from the OECD201 tests were analyzed. The resulting PCA is presented in Figure 2. The first axis is mainly driven by the interaction with the microalgae which is correlated with the surface charge values as it was already found in Chapter 3. This creates the bigger differentiation between the NMs. Two clusters and 3 detached NMs can be identified in the individual's graphic. The first group at the left is formed, as the same of the physicochemical PCA, by the 3 SiO2 NMs and CeO2<10nm.This group is characterized by a very low interaction with microalgae and negative surface charges. This phenomenon was already pointed out as an electrostatic attraction/repulsion principle with the microalgae cell wall that has a negative charged cell wall and responds to the DLVO principle [START_REF] Ma | The biophysicochemical interactions at the interfaces between nanoparticles and aquatic organisms: adsorption and internalization[END_REF]. The second group can be subdivided in one part with the two rutile and coated TiO2 NM103 and104, and another part with TiO2-NM102, TiO2-NM105 and CeO2<25nm. The NMs that are detached from the others are TiO2-NM100, TiO2-NM101 and ZnO-NM110. It is important to note that the data representing the NMs toxicity to algae (OECD201) does not correlate with the variables of NMs-algae interaction.

The groups identified in this analysis are similar to those found in the analysis of the NMs PC characteristics. This means that at least concerning the interaction with algae, the selected PC properties can be considered suitable to represent the nano-bio interaction. A similar analysis was made by (Hund-Rinke et al., 2018) that compared the grouping classification obtained with NMs PC data and ecotoxicological data, and found that some of the chosen PC parameters (solubility, reactivity, morphology) could be relevant to predict the toxicity towards microalgae (OECD test 201). In addition, considering the importance of the nano-bio interaction data in these kinds of analysis, (Schwirn and Völker, 2019) concluded that the attachment of NMs towards algae seems important for the prediction of adverse effects.

NMs-daphnid kinetics

The analysis between the NMs PC characteristics and the NMs uptake and elimination kinetics results is showed in Figure 3. The NMs distribution of this analysis if very different from the others. Two NMs outstand from the rest, SiO2-NM202 and ZnO-NM110, principally because they showed very different uptake/elimination behaviors than the others. SiO2-NM202 did not had an uptake at all in daphnids and ZnO-NM110 had a unique profile due to ZnO dissolution. For the rest, two clusters can be identified, one composed by CeO2<25nm and all TiO2 NMs except TiO2-NM101. And other composed by TiO2-NM101, CeO2<10nm, SiO2-NM200 and SiO2-NM204;

characterized by more negatively charged NMs and slower uptake rates to daphnia.

NMs with similar PC characteristics did not interact with the daphnids in a similar way, thus these PC parameters are not enough to explain the uptake and elimination behavior of NMs in daphnids. Additionally that the NMs PC measures in the media are not explicative of the real fate of NMs inside the organisms, since NMs may go through PC changes in daphnids digestive tubes due to the different pH conditions [START_REF] Davis | Development and application of a ratiometric nanosensor for measuring pH inside the gastrointestinal tract of zooplankton[END_REF] and that may lead to an increased local ion release that is not apparent from the solubility in the test media (Schwirn and Völker, 2019).

NMs-daphnid early effects

The differential gene expressions of 11 target genes at 24h of exposure to the NMs is analyzed with the PC characteristics and showed in Figure 4. In this analysis the first axis is driven by the transcriptomic parameters and the second axis is most driven by the NMs PC characteristics. In what concerns the grouping of NMs according to this analysis, a reappearing group is the one formed by TiO2-NM103 and TiO2-NM104, this time accompanied by TiO2-NM105. This group is characterized by general upregulation of genes in response to the NMs exposure and mostly positive surface charges in the media. Furthermore, one other, but more dispersed cluster can be identified, formed again by all three SiO2 NMs.

TiO2-NM101 is very different from the rest because of its extreme gene regulation results, the daphnids exposed to this NM showed the metallothionein gene expression highly upregulated and almost all the rest highly downregulated; which can be translated in a high toxic response from the organisms in comparison with the other NMs, which has been also observed in the OECD202 values for this NM that is the only one of the TiO2 with EC50 values under 100mg/L. ZnO-NM110 also presents a very different behavior from the others in this analysis and is the only other NMs presenting EC50 values under 100mg/L. Finally, no correlation could be stablished between the acute toxicity results of the OECD202 and the NMs PC characteristics nor the transcriptomics results for the exposure phase.

The differential expression of the same genes after 5 days of elimination were also analyzed with the NMs PC characteristics and showed in Figure 5. For the elimination part, TiO2-NM105 (mix anatase/rutile) has a completely different behavior that the one showed during the exposure phase where it displayed a similar behavior as the rutile NMs. Still both rutile and coated TiO2 NMs can be clustered together, having similar responses at modulating gene responses with an upregulation of genes and positive surface charges. Another group of NMs that show similarities can be formed by the TiO2-NM100 and CeO2<25nm. The SiO2 NM, as well as the rest of the TiO2 NMs are all of them very dispersed on the individuals' graphic, due to their different responses to the transcriptomic analysis for the elimination phase. The NMs that showed a very different behavior than the others are CeO2<10nm, that showed some upregulates gene expression during this phase and that has negative surface charge; and ZnO-NM110 that showed a different profile of genetic expressions with a high upregulated metallothionein and most of the other genes downregulated.

A final analysis was made only with the transcriptomic results from exposure and elimination of NMs to daphnids. The analysis is presented in Figure 6. It is important to note that the classification by similarities of NMs in this analysis is alike the other analysis that considered the NMs PC characteristics. This can be interpreted as a demonstration of the connection that some NMs characteristics have towards their early molecular effects in organisms, even if it is still difficult to relate which ones and how they work.

Interest of the NMs PC intrinsic and extrinsic characteristics along with the nano-bio interaction and early molecular effects for the classification of NMs

All along the different components of the analyses of this work, an important differentiation was raised for the two different crystalline forms of TiO2 that were almost systematically clustered in different groups. The structure of NMs is becoming more and more important as a descriptor for NMs toxicity and it has been demonstrated a significantly different toxicity between two crystalline forms of SiO2 (quartz and amorphous) [START_REF] Ghiazza | Does Vitreous Silica Contradict the Toxicity of the Crystalline Silica Paradigm?[END_REF], or between anatase and rutile forms of TiO2 (Clément et al., 2013).

Concerning the intrinsic and extrinsic PC characteristics, along the different analyses, the pristine size and the specific surface area as intrinsic along with the NMs zeta potential (extrinsic) were the most well representing descriptor variables for the NMs PC properties. The role of the pristine and media dependent NMs PC characteristics as descriptors for nanotoxicity is an emerging concept, but very important since many of the PC characteristics of NMs are "context dependent", affected by the surrounded media, the route of exposure, etc. (Lynch et al., 2014). This underlines the importance of including both the intrinsic and extrinsic NMs characteristics in grouping strategies.

The analysis approach of the present study is framed within the recommendations of ECHA to consider other NMs properties for grouping strategies and it highlights the interest of including nano-bio interaction and early molecular effects data to the grouping approaches of NMs.

The study of (Hund-Rinke et al., 2018) that used anatase and rutile forms of TiO2 NMs and that grouped them by their toxicity (EC50) towards algae, daphnids and fish embryos, did not found any significant difference to separate those two crystalline forms in their grouping proposal. Our analysis shows almost systematically that, between our studied NMs, rutile and anatase forms are very different in their way of interacting with algae and daphnids and found themselves in different groups. Additionally, the work of (Hund-Rinke et al., 2018), did not found any influence of the NMs surface charge in the ecotoxicological results, NMs with different zeta potentials presented similar EC50 for daphnids and fish embryos and in the algae media all of their NMs presented negative zeta potentials. On the contrary, in our study, the NMs surface charge presented a relevant correlation with the algae-NMs interactions, leading to classify differently the positively and negatively charged NMs.

The same study of (Hund-Rinke et al., 2018), tested a TiO2 NM (rutile) coated with AlO3 at 6% similar to the one used in our study, and they could also observe a lower toxicity associated with this NM in comparison to a similar NM without the coating. This can explain also the differences in the grouping positioning between the 2 rutile TiO2 tested NMs and the others, thus a rutile formed NM without the coating would be necessary in future studies to finish elucidating these grouping differences.

Conclusions

The purpose of this study was to analyze the possibility of stablishing groups of 12 metallic oxide NMs, using a large dataset of known and generated parameters in three categories: pristine PC characteristics, measures of NMs characterization in the different exposure media, and nano-bio interaction and toxicity towards 2 trophic level freshwater organisms. The analysis interweaves these 3 categories to NMs grouping, as it has already been presented as relevant for a better understanding of NMs hazards. In general, it was possible to form groups of NMs based on their PC properties or interaction and toxicity with the tested organisms. The PCA analysis has allowed to point out the groups and to refer to the explicative parameters behind the similar or different behaviors. The only phenomena that cannot correlate to the NMs PC properties was the uptake/elimination kinetics in daphnids.

The different PCA analysis showed some major trends and some group repartitions were recurring. The most repeated group of similar NMs was the couple TiO2-NM103 and TiO2-NM104, both rutile formed and coated. They showed similar correlations between their pristine characteristics and their fate in the tested media, as well as correlations between those two characteristics and their interaction with algae, daphnids' uptake and elimination kinetics and early molecular effects to exposure and elimination.

The SiO2 NMs showed to be very similar between each other in almost all analysis except for two analysis where the most different NMs was SiO2-NM202 that has displayed some different behaviors towards the gene expression responses during the elimination phase and that did not show any uptake in daphnids. Sometimes CeO2<10nm was also similar to the SiO2 NMs group, especially concerning the intrinsic and extrinsic PC characteristics and the algae-NMs interactions. This group consists in NMs that forms small agglomerates, negatively charged in their surface and showing low interactions with algae and slow to no uptake to daphnids. The two CeO2 NMs were very different and through all the analysis they were classified in different groups or all alone, demonstrating that the composition for these NMs is not an important grouping parameter. CeO2<25nm was placed often close to the anatase TiO2 group, especially similar to TiO2-NM102.

The TiO2-NM101 has shown to be the most different from the rest, by its PC properties (small pristine size) as well as for its higher toxicity showed in the regulatory tests and in the results from the transcriptomic analysis. ZnO-NM110 has also been frequently placed alone, since it has showed different uptake/elimination kinetics and toxic responses than the others.

Finally, it can be concluded that some NMs physicochemical characteristics (size, surface charge, crystalline structure) can determine the formation of similar NMs clusters that interact with or affect the organisms in a similar way. This highlights the importance of considering the nano-bio interaction data for grouping strategies. Further studies should verify these correlations and, since the diversity of studied NMs for this work was anyway limited, different NMs should be studied to enrich this dataset.

Acknowledgements:

The authors thank the support of the French Ministry for the Ecological and Inclusive Transition. A special recognition is given to Cléo Tebby for the assistance with the statistical analysis.

Main conclusions

To answer this chapter's presented questions, various principal component analyses were made allowing to cluster some NMs together and it can be concluded that:

• Clusters of NMs with similar PC characteristics were determined. The main variables that allow this classification are the NMs surface charge (media dependent)

and the NMs pristine size and specific surface area (intrinsic properties). The resulting NMs classified inside a cluster, have also concordance with the composition and crystalline form or coating characteristics.

• The NMs clusters formed by similarities of PC characteristics and nano-bio interaction with organisms were similar to those only based on PC parameters, having the main influence of the NMs surface charge (media dependent) and the nano-bio interactions (algae-NMs interactions or early molecular effects on daphnids). But the NMs clusters formed for the uptake and elimination behaviors on daphnids were no similar to the others, which can be explained by the different NMs PC characteristics inside de organisms.

• Based solely on the mRNA expression differences found on daphnids, the NMs can be clustered in a similar way that the one that includes their PC characteristics, which

shows that some connections can be made.

• The nano-bio interaction and NMs early molecular effects can be of high relevance in NMs grouping strategies, since the ultimate finality of grouping is to determine the environmental risk of NMs, and since only NMs physicochemical characteristics are not enough to explain the toxic effects; it becomes essential to consider the interactions and effects of NMs towards biota for NMs grouping approaches. This study has showed that the inclusion of these parameters can change the way NMs have been classified by other grouping strategies, thus these strategies can only be enriched by the inclusion of more bio-related parameters.

Conclusions and Perspectives

The present work, conceived as a systematically and innovative study, has sought to characterize the interaction of NMs with biological systems, highlighting them as complementary criteria of the classic toxic tests for NMs grouping purposes. The findings of this thesis have contributed to a better understanding of the phenomena of interaction, uptake and elimination behaviors and early molecular effects of NMs for freshwater organisms belonging to two trophic levels. The main conclusions of this work are summed up as follows:

A short time contact test and analysis by flow cytometry has been successfully developed for the characterization of the interaction between NMs and freshwater microalgae.

This almost instantaneous interaction was found to be dependent on the type of organisms and the type of NMs. The differences between the NMs interactions have shown to be related to the NMs chemical composition, zeta potential, and crystalline phase.

The uptake and elimination behaviors of the NMs were determined in the microcrustacean D. magna and a toxicokinetic model of one compartment has proven to be adequate to describe them. NMs dissolution and hydrodynamic size in the media have shown to play a key role in their uptake behavior and the elimination is mainly promoted by the food ingestion. The obtained uptake and elimination rates allowed to cluster the NMs according to low, intermediate or fast rates, but no correlations between the clustered NMs and their physicochemical properties were found.

Transcriptomic analysis of the NMs early molecular toxic effects in D. magna, have evidenced alterations in the expression of genes related to oxidative stress defense, repair of damaged DNA, restoration of misfolded proteins, metal detoxication and endocytosis at a cellular level; and developing, molting, reproduction and digestion at an individual level.

Genetic expression of both cellular and individual processes showed alterations during the NMs exposure, and mainly those of reproduction and metal biding were still highly impacted after elimination showing a general resilience capacity of the organisms. NMs crystalline structure or coating seemed to be involved in the toxic effects' differences.

NMs were clustered together according to the similarities of their physicochemical characteristics and the interrelationship of those characteristics with the nano-bio interactions.

Recurrent group's conformations were found with different descriptors, meaning that there are some NMs with similar characteristics that interact with organisms in a similar way. However, no particular descriptor was found directly related to these similarities.

Resuming the principal findings of this work, the influence of the different NMs characteristics in their studied interactions with organisms is showed in Figure 40. The strong relationships are marked with a big star. Small stars represent relationships where more than one parameter applied at the same time and clear identification was not possible. Small circles designate the characteristics that may seemed to have an influence but did not applied for the entire set of NMs. Dashes are placed where the parameters showed no apparent influence. In general, it can be concluded that the physicochemical characteristics of NMs (intrinsic and media-dependent) are very important in their interaction with aquatic organisms for grouping approaches, even if no clear direct dependence between one specific NMs property and a specific effect or interaction behavior could have been verified, some recurrent tendencies suggest relationships that are still to be further studied. Based on the findings of this thesis, it can be concluded that the analysis of the NMs interactions with biological systems should be incorporated in risk assessment studies and grouping approaches. Nevertheless, it is still a complex task, due to the multi-variability of endpoints and the parameters dependencies on the type of organisms, the type of NMs and the media/environment characteristics.

A limitation of these kind of large systematic studies is that, even if the chosen set of NMs with different physicochemical characteristics is very useful for the research of the relationships between the NMs characteristics and the interaction/effects in organisms, there is more than one parameter that varies between the NMs. Then, the cause-effect correlations became more complex. On the other hand, it would be extremely difficult to compose a set of NMs that allows to analyze many parameters having each one in ceteris paribus conditions.

Concerning the set of chosen NMs, it would be interesting to enlarge the study to NMs with other physicochemical properties as carbon based NMs, other soluble NMs as silver and copper, QDs, nanocomposites, etc. Additionally, some of the protocols of this thesis can be suitable for studying other particulate-formed pollutants as submicronic polymers.

The algae-NMs interaction results and the toxic effects results from the OECD 201 test, did not present any correlation, suggesting that the measured NMs attachment to microalgae is not related to their toxicity. However, the TEM images showed that the NMs agglomerates seemed to impact the microalgae cell wall integrity. To deepen into this, dying the cells can be used to verify the damages to their cell walls, and the use of fluorescence microscopy and flow cytometry analysis can facilitate the verification and quantification of the impacted population, as shown by [START_REF] Machado | Development of a short-term assay based on the evaluation of the plasma membrane integrity of the alga Pseudokirchneriella subcapitata[END_REF]. Likewise, the analysis of other algae species with different cell wall properties and extracellular polymeric substances can deepen the knowledge of NMs attachment and interactions with the algae cell walls. Hydrophobicity can also be a relevant parameter for the cellular adhesion with microalgae; some preliminary results presented in Chapter 3 showed indices of its relation with the differences in the nano-bio interaction, further investigation is needed.

To better represent the freshwater environment characteristics, the same nano-bio interaction analyses presented in this work could be done in the future, adding natural organic matter (NOM), and the comparison with the present results would be informative about the role of NOM in the behavior of NMs in the media and its impact on the exposure and effects towards aquatic organisms. Existing studies show that the presence of organic matter alter the surface properties, stability and bioavailability of NMs, thus impacting their interaction with organisms as algae (Lynch et al., 2014) [START_REF] Monikh | Do the joint effects of size, shape and ecocorona influence the attachment and physical eco(cyto)toxicity of nanoparticles to algae?[END_REF], and the uptake and elimination behavior in daphnids [START_REF] Nasser | Updating traditional regulatory tests for use with novel materials: Nanomaterial toxicity testing with Daphnia magna[END_REF]. The TEM images of algae exposed to the set of NMs and NOM presented in Annex 2, seem to show less NMs agglomerates attached to the algae cells.

Further studies using the here presented short time contact test and flow cytometry analysis can confirm and quantify this difference.

Concerning the NMs interaction with the primary consumers, there are still some gaps that need to be filled to fully understand the NMs uptake and elimination behaviors. Special attention is required to the NMs physicochemical characterization inside the organisms' digestive tubes conditions.

Another technique that can be useful for a more detailed determination of the uptake and elimination mechanisms in daphnids is the single particle inductively coupled plasma mass spectrometry (sp-ICP-MS). Preliminary results have seemed to show a different NM size distribution in the media at the beginning and at the end of the uptake phase, which could confirm the concept that the NMs uptake is affected by the size of agglomerates filtered by the organisms.

The nano-bio interactions of the primary producers and a primary consumer were studied in this work by direct exposure of NMs in the media. To complete the characterization, the inclusion of both species in a trophic transfer analysis can be useful.

The results of the qPCR analysis after the elimination phase, showed that the vitellogenin gene expression remained altered for almost all the tested NMs, meaning that the daphnids reproduction process can be impacted even long after the exposure. To deepen into the long-term effects of NMs exposure, transgenerational studies can inform about the possible impacts of an exposed population and its further generations. Studies of transgenerational effects of NMs in daphnids have begun to appear as the ones of (Hartmann et al., 2019) and (Ellis et al., 2021).

To finally answer the question of whether it is possible to translate the NMs physicochemical characteristics into the NMs biological effects, it is still necessary to go further in the study of descriptors and refining the analysis to parameter-by-parameter. Starting with the NMs characteristics identified in this thesis as having an influence on the outcomes of the NMs interactions with biota, more precise and refined analyses are needed for a better understanding of each one. The grouping strategies, based on NMs characteristics as descriptors for their toxic effects and biological-related interactions, remain in ongoing development. Work is still needed to find fully reliable approaches where the NMs hazards could be described without additional testing. Until then, NMs case-by-case studies will continue to be required for risk assessment determinations. -Posterior Predictive Check (PPC)

The PPC shows the observed values against their corresponding estimated predictions (black dots), along with their 95% credible interval (vertical segments). Ideally observations and predictions should coincide and would be expected as black dots along the first bisector y = x (plain black line). The 95% credible intervals are colored in green if they overlap this line

1 .

 1 Introduction and context ............................................................................................. 2. Nanomaterials characteristics, nano-bio interactions and early molecular effects as elements for grouping strategies (in prep.) ........................................................................ 3. Main conclusions ........................................................................................................ Conclusions and Perspectives ........................................................................... References ......................................................................................................... Annex 1: Media compositions ........................................................................... Annex 2: TEM images algae-NMs and NOM .................................................. Annex 3: Supplementary modelling results ...................................................... Annex 4: General data matrix ...........................................................................

Figure 1 :Figure 2 :

 12 Figure 1: Schematic diagram of the basic classification of nanomaterials (Andrea Rivero Arze) .................................................................................................................................................. 11 Figure 2: General structure of the NMs flow throughout their life cycle(Sun et al., 2014). .. 15

Figure 3 :Figure 4 :

 34 Figure 3: Estimated global mass flow of ENMs (in metric tons per year) from production to disposal or release, considering high production and release estimates as of 2010 (Keller and Lazareva, 2014). ....................................................................................................................... 16 Figure 4: Modeled and analytical concentrations of NMs in surface waters. Green boxes show the range (and the arithmetic mean on the log scale) of modeled results. Yellow boxes show measured concentrations and the orange boxes combine measurements and modeling[START_REF] Gottschalk | Environmental concentrations of engineered nanomaterials: Review of modeling and analytical studies[END_REF]. Sources of data 1.[START_REF] Mueller | Exposure Modeling of Engineered Nanoparticles in the Environment[END_REF]; 2.[START_REF] Gottschalk | Modeled Environmental Concentrations of Engineered Nanomaterials (TiO2, ZnO, Ag, CNT, Fullerenes) for Different Regions[END_REF]; 3.(O'Brien and Cummins, 2010); 4.[START_REF] Musee | Simulated environmental risk estimation of engineered nanomaterials: A case of cosmetics in Johannesburg City[END_REF]; 5.[START_REF] Gottschalk | Engineered nanomaterials in rivers -Exposure scenarios for Switzerland at high spatial and temporal resolution[END_REF] 6.(A. C. Johnson et al., 2011); 7.(Neal et al., 2011) and 8.(A. Johnson et al., 2011). ........................... 17

Figure 5 :Figure 6 :Figure 7 :Figure 8 :Figure 9 :Figure 12 :Figure 13 :Figure 14 :Figure 15 :Figure 18 :Figure 19 :Figure 20 :Figure 21 :Figure 22 :Figure 23 : 57 Figure 25 :Figure 27 :Figure 28 :Figure 29 :Figure 30 :Figure 31 :

 567891213141518192021222357252728293031 Figure 5: Distribution and processes of NMs in the environmental compartments (Lespes et al., 2020). .................................................................................................................................. 18 Figure 6: Schematic overview of mechanisms involved in toxicity alteration of environmentally transformed NMs (Zhang et al., 2018) .......................................................... 19 Figure 7: The importance of NMs characterization and behavior in nano-ecotoxicity studies in the last 20 years by number of publications. Modified from (Corsi et al., 2021). ................... 25 Figure 8: Examples of the main NMs physical interactions with microalgae and microcrustaceans. Source of the images: (Manier et al., 2013) and non-published photos from N. Manier. ................................................................................................................................ 28 Figure 9: A. Optical microscopy image at 40x objective of a culture of R. subcapitata at INERIS facilities. B. and C. TEM images of longitudinal section and cross-section respectively; chloroplast (c), nucleus (n), starch grain (s), pyrenoid (star); scale 1µm (Krienitz et al., 2011). .................................................................................................................................................. 34 Figure 10: A. Optical microscopy image at 40x objective of a culture of D. subspicatus at INERIS facilities. B. Microphotograph of Desmodesmus subspicatus strain TAU-MAC 2810 under light microscopy. Scale 10 µm (Lortou and Gkelis, 2019). ........................................... 35 Figure 11: A. Optical microscopy image at 40x objective of a culture of C. vulgaris at INERIS facilities. B. Electron micrograph of a cell of C. vulgaris in longitudinal section. Cell wall (cw), cup shaped chloroplast (ch), thylakoids (t), starch grains or leucoplasts (st), nucleus (n), nucleolus (nl), mitochondria (m). Scale 1 µm (Tomaselli, 2003). ........................................... 36 Figure 12: Different phases of daughter cell-wall formation in Chlorella vulgaris: (a) early cell-growth phase; (b) late cell-growth phase; (c) chloroplast dividing phase; (d) early protoplast dividing phase; (e) late protoplast dividing phase; (f) daughter cells maturation phase and (g) hatching phase (Yamamoto et al., 2005). ................................................................................. 36 Figure 13: The functional anatomy of Daphnia magna. Modified from (Ebert, 2005). ......... 38 Figure 14: Filtering and ingestion rates of algal cells (Chlamydamonas reinhardtii) by Daphnia magna as a function of algal concentration. Although the filtering rate falls off sharply with

Figure 32 :Figure 33 :Figure 34 :Figure 35 :Figure 36 :Figure 37 :Figure 38 :Figure 39 :Figure 40 :Figure 41 :Figure 42 :Figure 43 :Figure 44 :Figure 45 :Figure 46 :Figure 47 :

 32333435363738394041424344454647 Figure 32: Schematic description of the analysis daphnids-NMs interaction for the uptake and elimination behaviors (Andrea Rivero Arze) ........................................................................... 90 Figure 33: Measured and predicted NMs concentrations in the organism ............................ 103 Figure 34: Percentage of dissolved Zn ions in relation to the Zn concentration of the exposure media. Error bars represent the standard deviation from the triplicates. ................................ 104 Figure 35: Binocular loupe images (20x) of the entire body of D. magna at the end of the uptake phase (24h at 1mg/L). ............................................................................................................. 105 Figure 36: Binocular loupe images (90x) of the heads of D. magna at the end of the uptake phase (24h at 1mg/L). ............................................................................................................. 106 Figure 37: Binocular loupe images (90x) of the carapace surfaces of D. magna at the end of the uptake phase (24h at 1mg/L). ........................................................................................... 107 Figure 38: Schematic description of the analysis of the effects on the genetic expression of D. magna exposed to NMs (Andrea Rivero Arze) ...................................................................... 110 Figure 39: Schematic description of the analysis of grouping possibilities based on NMs physicochemical characteristics and nano-bio interactions (Andrea Rivero Arze) ............... 136 Figure 40: Influence of the different NMs physicochemical characteristics in the interactions between NMs and the studied organisms. Big stars designate strong influences, small stars represent influences where the parameters could not have been clearly separate, and circles designate possible influences. (Andrea Rivero Arze) ............................................................ 160 Figure 41: TEM images of algae-NMs interaction for R. subcapitata and the 5 tested NMs with 50 mg/L of NOM. ................................................................................................................... 181 Figure 42: TEM images of algae-NMs interaction for D. subspicatus and the 5 tested NMs with 50 mg/L of NOM. ................................................................................................................... 182 Figure 43: Modelling resulting BCFk distributions .............................................................. 184 Figure 44: Posterior Predictive Check .................................................................................. 185 Figure 45: Priors and posteriors ............................................................................................ 187 Figure 46: Correlation between parameters .......................................................................... 188 Figure 47: Correlations between BCFk and Kuw or Kee. Error bars correspond to the 2.5% and 97.5% quantiles for the BCFk, kuw and kee values. ....................................................... 189

Table 4 :

 4 . .......................... 30 Table 2. Values of 48h EC50 and LC50 of some metal oxide NMs on daphnids. ..................... 31 Table 3: Main characteristics of the set of selected NMs ........................................................ 43 Preparation volumes of algae, media and NMs intermediate suspension. ................ 50 Table 5: Limits of quantification for the ICP-OES analysis of organisms' samples and water samples. .................................................................................................................................... 60 Table 6: Number of R. subcapitata cells in control population for the interaction kinetic tests and the calculation of the coefficient of variation. ................................................................... 79 Table 7: Percentage difference of the algae-NMs granularity modification between the three tested kinetic times. .................................................................................................................. 80 Table 8: Acute toxicity results EC10, EC25 and EC50 of the OECD 201 test of growth inhibition on freshwater algae. n.a. = Data not available. Data obtained under and belonging to the NANoREG2 project. ................................................................................................................ 87 Table 9: Lefevre Czarda Media (LC) composition. ............................................................... 179 Table 10: Algae OECD Growth Media ................................................................................. 179 Table 11: M4 composition ..................................................................................................... 180 Table 12: ISO media composition ......................................................................................... 180 Table 13: Additional results from modeling. Number or iterations for each NMs, stochastic variability around the trend average (σp) and potential scale reduction factor (PSRF) ......... 183 Table 14: General data matrix used for the principal component analyses for NMs clustering ................................................................................................................................................ 190

  Dans une seconde partie, l'interaction (accumulation, élimination) des NM avec le microcrustacé d'eau douce (D. magna) a été étudiée. D'une façon générale, les NM ont montré des comportements d'absorption rapide pendant les premières heures d'exposition atteignant un plateau vers 24h. Les comportements d'élimination ont présenté une perte rapide par excrétion au cours des premières heures mais des concentrations résiduelles ont pu être retrouvées dans les organismes après 5 jours d'élimination. Un modèle toxicocinétique a été appliqué et les taux d'absorption et d'élimination ainsi que les facteurs dynamiques de bioconcentration ont été définis.

  NMs and biological systems, as complementary parameters to the classic toxic effects studies, by working with representative organisms of the two basic levels of a freshwater ecosystem's trophic chain and a set of metallic oxide NMs with different physicochemical properties; placing the study in the context of classification of NMs and studying the evolution of these parameters for NMs grouping purposes.

Figure 1 :

 1 Figure 1: Schematic diagram of the basic classification of nanomaterials (Andrea Rivero Arze)

Figure 2 :

 2 Figure 2: General structure of the NMs flow throughout their life cycle (Sun et al., 2014).

Figure 3 :

 3 Figure 3: Estimated global mass flow of ENMs (in metric tons per year) from production to disposal or release, considering high production and release estimates as of 2010(Keller and Lazareva, 2014).

Figure 4 :

 4 Figure 4: Modeled and analytical concentrations of NMs in surface waters. Green boxes show the range (and the arithmetic mean on the log scale) of modeled results. Yellow boxes show measured concentrations and the orange boxes combine measurements and modeling (Gottschalk et al., 2013). Sources of data 1.(Mueller and Nowack, 2008); 2.(Gottschalk et al., 2009); 3.(O'Brien and Cummins, 2010); 4.(Musee, 2011); 5.(Gottschalk et al., 2011); 6.(A. C. Johnson et al., 2011); 7.(Neal et al., 2011) and 8.(A. Johnson et al., 2011).

Figure 5 :

 5 Figure 5: Distribution and processes of NMs in the environmental compartments (Lespes et al., 2020).

Figure 6 :

 6 Figure 6: Schematic overview of mechanisms involved in toxicity alteration of environmentally transformed NMs[START_REF] Zhang | The effects and the potential mechanism of environmental transformation of metal nanoparticles on their toxicity in organisms[END_REF]

Figure 7 :

 7 Figure 7: The importance of NMs characterization and behavior in nano-ecotoxicity studies in the last 20 years by number of publications. Modified from[START_REF] Corsi | Eco-Interactions of Engineered Nanomaterials in the Marine Environment: Towards an Eco-Design Framework[END_REF].

Figure 8 :

 8 Figure 8: Examples of the main NMs physical interactions with microalgae and microcrustaceans. Source of the images: (Manier et al., 2013) and non-published photos from N. Manier.

between 1 .

 1 6 and 4.4 μm. Each cell has a single parietal chloroplast (Figure9)[START_REF] Padmakumar | First Identification of the Chlorophyte Algae Pseudokirchneriella subcapitata (Korshikov) Hindák in Lake Waters of India[END_REF] Tharavathy, 2020) (Huarachi-Olivera et al., 2019).

Figure 9 :

 9 Figure 9: A. Optical microscopy image at 40x objective of a culture of R. subcapitata at INERIS facilities. B. and C. TEM images of longitudinal section and cross-section respectively; chloroplast (c), nucleus (n), starch grain (s), pyrenoid (star); scale 1µm (Krienitz et al., 2011).

Figure 10 :

 10 Figure 10: A. Optical microscopy image at 40x objective of a culture of D. subspicatus at INERIS facilities. B. Microphotograph of Desmodesmus subspicatus strain TAU-MAC 2810 under light microscopy. Scale 10 µm (Lortou and Gkelis, 2019).

Figure 11 :

 11 Figure 11: A. Optical microscopy image at 40x objective of a culture of C. vulgaris at INERIS facilities. B.Electron micrograph of a cell of C. vulgaris in longitudinal section. Cell wall (cw), cup shaped chloroplast (ch), thylakoids (t), starch grains or leucoplasts (st), nucleus (n), nucleolus (nl), mitochondria (m). Scale 1 µm[START_REF] Tomaselli | The Microalgal Cell, in: Handbook of Microalgal Culture[END_REF].

Figure 12 :

 12 Figure 12: Different phases of daughter cell-wall formation in Chlorella vulgaris: (a) early cell-growth phase; (b) late cell-growth phase; (c) chloroplast dividing phase; (d) early protoplast dividing phase; (e) late protoplast dividing phase; (f) daughter cells maturation phase and (g) hatching phase[START_REF] Yamamoto | Late type of daughter cell wall synthesis in one of the Chlorellaceae, Parachlorella kessleri (Chlorophyta, Trebouxiophyceae)[END_REF].

Figure 13 :

 13 Figure 13: The functional anatomy of Daphnia magna. Modified from[START_REF] Ebert | Introduction to Daphnia Biology, Ecology, Epidemiology, and Evolution of Parasitism in Daphnia[END_REF].

Figure 14 :

 14 Figure 14: Filtering and ingestion rates of algal cells (Chlamydamonas reinhardtii) by Daphnia magna as a function of algal concentration. Although the filtering rate falls off sharply with increased concentration, the ingestion rate increases up to a point and then becomes constant[START_REF] Dodds | 18 -Behavior and Interactions among Microorganisms and Invertebrates[END_REF].

Figure 15 :

 15 Figure 15: Life cycle of sexual and asexual reproduction cycles on Daphnia magna[START_REF] Ebert | Introduction to Daphnia Biology, Ecology, Epidemiology, and Evolution of Parasitism in Daphnia[END_REF].

Figure 16 :

 16 Figure 16: A. Bottles of NMs in powder form provided by the JRC. B. Stock suspension of TiO2 NMs at 2.56 g/L. (Photos: Rivero Arze, A.).

  .D.).

Figure 17 :

 17 Figure 17: A. Algae inoculum ready to refrigerate. B. Algae mother strain culture of 1 week old. C. Setup for the algae culture with the horticultural lightning and the orbital rotation table. D. Algae pre-cultures. (Photos: Rivero Arze, A.)

Figure 18 :

 18 Figure 18: Equipment for the algae-NMs short-time contact test. A. Supporting equipment. B. Zoom of the vials support. C. Glass vial containing the magnetic stirring rod and one of the NMs control samples. (Photos Rivero Arze, A.).

  a sample of 200µL was taken from each vial and placed into a 96 well plate to be analyzed by the flow cytometer. The plate setup is presented in Figure19.

Figure 19 :

 19 Figure 19: Algae-NMs plate setup for flow cytometry analysis.

  The fluidic system is responsible for transporting cells individually through pressurized lines containing sheath fluid to the interrogation point, where the laser intersects with the sample. The optical system is responsible for illumination and light collection, and it is comprised of excitation lasers, lenses and filters. The lasers ensure that cells in the interrogation point are excited with uniform light of a specific wavelength. As a cell passes through the laser, it emits fluorescence and scattered laser light at all angles. The collection lenses and filters serve to separate and direct the specific wavelengths of fluorescence and scattered laser light to the appropriate detectors. These detectors capture and convert the emitted fluorescence and scattered laser lights, into a photocurrent and passes it to the electronics system to be digitized and processed for subsequent analysis ([START_REF] Bioquest | Fundamentals of Flow Cytometry[END_REF].

Figure 20 :

 20 Figure 20: Schematic of a common flow cytometer, illustrating the fluidic, optical, and electronic systems (AAT[START_REF] Bioquest | Fundamentals of Flow Cytometry[END_REF]

Figure 21 :

 21 Figure 21: Example of the analysis of the percentage of algae with higher complexity referring to a control population. Up left: control TiO2-NM101 sample. Up right: algae control population sample of Raphidocelis subcapitata. Down left: interaction sample R. subcapitata + NM101 at 25 mg/L.

Figure 22 :

 22 Figure 22: Photos of the preparation phase for the exposition population. A. Mothers culture aquariums. B. Mothers with neonates. C. Filtration of neonates. D. Synchronized growth of exposure population. (Photos: Rivero Arze, A.).

Figure 23 :

 23 Figure 23: Experimental design of NMs exposure and elimination phases for D. magna.

Figure 24 :

 24 Figure 24: A. Set up of the uptake and elimination experiment. B. Start of the uptake phase with 50 daphnids in the container. C. Sample of 25 daphnids after 24h of exposure to a TiO2 NM, the guts are visually fulfilled with NMs. D. Samples covered in aluminum wrapping, destined for transcriptomic analysis, ready to be frozen. (Photos: Rivero Arze, A.).

Figure 25 :

 25 Figure 25: Schematic operating mode of the ICP-OES analysis (Neale, 2020).

Figure 26 :

 26 Figure 26: A. ICP-OES device at INERIS facilities. B. Sample rack of the ICP-OES. (Photos: Rivero Arze, A.).

Figure 27 :

 27 Figure 27: Schematic description of the main characteristics of the algae-NMs interaction issue in the present study (Andrea Rivero Arze)

Figure 28 :

 28 Figure 28: Photos of the Dark field microscopy analysis on the hydrophobic collector T0 for determining algae-NMs hydrophobicity using D. subspicatus. Right: NM103. Left: NM101. (Photos: Rivero Arze, A.).

Figure 29 :

 29 Figure 29: NMs hydrophobicity vs. percentage of modified granularity by the algae-NMs interaction.

Figure 30 :

 30 Figure 30: TEM images of algae-NMs interaction for R. subcapitata and the 5 tested NMs.

Fig. 30

 30 Fig.30.F. shows the NMs penetrating the external layers of the cell wall. An heteroagglomerate of algae-NMs is observable in Fig.30.D., which can be assigned to the high complexity dots found by flow cytometry.

Figure 31 :

 31 Figure 31: TEM images of algae-NMs interaction for D. subspicatus and the 5 tested NMs.

Figure 32 :

 32 Figure 32: Schematic description of the analysis daphnids-NMs interaction for the uptake and elimination behaviors (Andrea Rivero Arze)

Figure 34 :

 34 Figure 34: Percentage of dissolved Zn ions in relation to the Zn concentration of the exposure media. Error bars represent the standard deviation from the triplicates.

Figure 35 :

 35 Figure 35: Binocular loupe images (20x) of the entire body of D. magna at the end of the uptake phase (24h at 1mg/L).

Figure 36 :

 36 Figure 36: Binocular loupe images (90x) of the heads of D. magna at the end of the uptake phase (24h at 1mg/L).

Figure 37 :

 37 Figure 37: Binocular loupe images (90x) of the carapace surfaces of D. magna at the end of the uptake phase (24h at 1mg/L).

Figure 38 :

 38 Figure 38: Schematic description of the analysis of the effects on the genetic expression of D. magna exposed to NMs (Andrea Rivero Arze)

  chosen metallic oxide NMs are considered as reference NMs as they are well known and characterized, mostly recovered from the JRC Nanomaterial Repository. The set is composed by 6 TiO2 NMs (NM100, NM101, NM102, NM103, NM104 and NM105), 3 SiO2 NMs (NM200, NM202 and NM204), 1 ZnO NMs (NM110) and 2 CeO2 NMs named in relation to their diameter size as CeO2<10nm (NanoBYK ® ) and CeO2 <25nm (Sigma Aldrich nanoCeO2). The description, preparation protocols and main characteristics of the NMs are detailed in a precedent work (Rivero Arze et al., 2020). NMs stock suspensions were prepared at 1 g/L in ultrapure water and added directly to the test containers with ISO media to reach the exposure concentration of 1 mg/L. Stock and exposure suspensions were characterized (size distribution and zeta potential) by Dynamic Light Scattering (DLS; NanoZS, Malvern Instruments ® ).

Figure 1 .

 1 Figure 1. Mortality of exposed organisms. a) At the end of the uptake phase (24h). b) At the end of the elimination phase (5 days). Statistical difference is marked with an *.

Figure 2 .

 2 Figure 2. Expressions of upregulated (positive values) and downregulated (negative values) mRNA level expressions in D. magna exposed to different NMs at the a) uptake and b) elimination phases. Only the values significantly different from control are presented. Data is expressed as log10 of the folding value.

Figure 39 :

 39 Figure 39: Schematic description of the analysis of grouping possibilities based on NMs physicochemical characteristics and nano-bio interactions (Andrea Rivero Arze)

  set consists in 6 TiO2 (NM100, NM101, NM102, NM103, NM104, NM105) (European Commission Joint Research Centre, 2014); 1 ZnO (NM110) (European Commission Joint Research Centre, 2012); 3 SiO2 (NM200, NM202, NM204) (European Commission Joint Research Centre, 2013) and 2 CeO2 named in relation to their diameter size as CeO2<10nm (NanoBYK ® ) and CeO2 <25nm (Sigma Aldrich nanoCeO2).

  samples were analyzed using a CyAn™ ADP High-Performance Flow Cytometer (Beckman coulter), where the fluorescence (FL1 Log) and the granularity or cell complexity (side scattering logarithm, SS Log) were measured. The displacement of the cytogram outline of the test samples (algae + NMs) was compared to the algae control population profile to find the percentage of algae with modified granularity. The data taken for the present work correspond to the percentages at the middle of the dose-response profile of each algae species (Int.Raphi, Int.Desmo and Int.Chlo).

Figure 1 .

 1 Figure 1. PCA analysis of the NMs physicochemical characteristics. Pist.size = Pristine size; SSA = Specific surface area; Dissol = Dissolution, ROS = reactive oxygen species generation; Size.stock = hydrodynamic diameter in ultrapure water; Size.algae = hydrodynamic diameter in algae media; Size.ISO = hydrodynamic diameter in daphnids media; Zpot.stock = surface charge in ultrapure water; Zpot.algae = surface charge in algae media; Zpot.ISO = surface charge in daphnids media.

Figure 2 .

 2 Figure 2. PCA analysis of the NMs physicochemical characteristics and the interaction with microalgae. Pist.size = Pristine size; SSA = Specific surface area; Dissol = Dissolution, ROS = reactive oxygen species generation; Size.stock = hydrodynamic diameter in ultrapure water; Size.algae = hydrodynamic diameter in algae media; Zpot.stock = surface charge in ultrapure water; Zpot.algae = surface charge in algae media, Int.Raphi = NMs interaction with Raphidocellis supcapitata; Int.desmo = NMs interaction with Desmodesmus subspicatus; Int.Chlo = NMs interaction with Chlorella vulgaris; OECD201 = EC50 results for the OECD 201 test.

Figure 3 .

 3 Figure 3. PCA of the NMs PC characteristics and the uptake and elimination kinetics in D.magna. Pist.size = Pristine size; SSA = Specific surface area; Dissol = Dissolution, ROS = reactive oxygen species generation; Size.stock = hydrodynamic diameter in ultrapure water; Size.ISO = hydrodynamic diameter in daphnids media; Zpot.stock = surface charge in ultrapure water; Zpot.ISO = surface charge in daphnids media; Up24h = NMs body burden at 24h of exposure; Elim120h = NMs body burden at 5 days of elimination; Kuw = NMs uptake rate; Kee = NMs elimination rate; BCF = NMs kinetic bioconcentration factor.

Figure 4 .

 4 Figure 4. PCA of the NMs PC characteristics and the differential gene expressions of 11 genes at 24h of exposure. Pist.size = Pristine size; SSA = Specific surface area; Dissol = Dissolution, ROS = reactive oxygen species generation; Size.stock = hydrodynamic diameter in ultrapure water; Size.ISO = hydrodynamic diameter in daphnids media; Zpot.stock = surface charge in ultrapure water; Zpot.ISO = surface charge in daphnids media; sod =superoxide dismutase; gst = glutathione S transferase; cyp = cytochrome P450; hsp = heat shock protein; mt = metallothionein; rev = DNA repair gene; flot = flotillin; jhe = juvenile hormone esterase; ecr = ecdysone receptor A; vtg = vitellogenin; amy = amylase; up = transcriptomic analysis at 24h of exposure.

Figure 5 .

 5 Figure 5. PCA of the NMs PC characteristics and the differential gene expressions of 11 genes at 5 days of elimination. Pist.size = Pristine size; SSA = Specific surface area; Dissol = Dissolution, ROS = reactive oxygen species generation; Size.stock = hydrodynamic diameter in ultrapure water; Size.ISO = hydrodynamic diameter in daphnids media; Zpot.stock = surface charge in ultrapure water; Zpot.ISO = surface charge in daphnids media; sod =superoxide dismutase; gst = glutathione S transferase; cyp = cytochrome P450; hsp = heat shock protein; mt = metallothionein; rev = DNA repair gene; flot = flotillin; jhe = juvenile hormone esterase; ecr = ecdysone receptor A; vtg = vitellogenin; amy = amylase; elim = transcriptomic analysis at 5 days of elimination.

Figure 6 .

 6 Figure 6. PCA of the NMs transcriptomic responses to the exposure and elimination of 11 target genes. sod =superoxide dismutase; gst = glutathione S transferase; cyp = cytochrome P450; hsp = heat shock protein; mt = metallothionein; rev = DNA repair gene; flot = flotillin; jhe = juvenile hormone esterase; ecr = ecdysone receptor A; vtg = vitellogenin; amy = amylase; up = transcriptomic analysis at 24h of exposure; elim = transcriptomic analysis at 5 days of elimination.

Figure 40 :

 40 Figure 40: Influence of the different NMs physicochemical characteristics in the interactions between NMs and the studied organisms. Big stars designate strong influences, small stars represent influences where the parameters could not have been clearly separate, and circles designate possible influences. (Andrea Rivero Arze)

Figure 42 :

 42 Figure 42: TEM images of algae-NMs interaction for D. subspicatus and the 5 tested NMs with 50 mg/L of NOM.

Figure 44 :

 44 Figure 44: Posterior Predictive Check

Figure 45 :Figure 46 : 4 :

 45464 Figure 45: Priors and posteriors

Table 2 .

 2 Values of 48h EC50 and LC50 of some metal oxide NMs on daphnids.

	NMs	Size (nm)	Species	Performances (mg/L)	Reference
		50	D. magna	EC50: 20.08	(Erdinçmer and Sponza, 2020)
	CeO2	8	D. similis	EC50: 0.26	(Artells et al., 2013)
		8	D. pulex	EC50: 91.79	(Artells et al., 2013)
		10 -20	D. magna	EC50: 148.87	(Han et al., 2009)
	SiO2	10 -20	D. magna	EC50: 10.01	(Erdinçmer and Sponza, 2020)
		20 -30	D. magna	LC50: 48.86	(Shariati et al., 2020)
		≤ 20	D. magna	EC50: 35.3	(Zhu et al., 2009)
	TiO2	21	D. magna	EC50: 19.3	(Amiano et al., 2012)
		19	D. magna	EC50: ˃100	(Hund-Rinke et al., 2018)
		10 -15	D. magna	EC50: 2.0	(Muna et al., 2019)
		20	D. magna	EC50: 0.62	(Zhu et al., 2009)
		70	D. magna	EC50: 2.6 ± 1.04	(Blinova et al., 2010)
	ZnO	41 42	D. magna D. magna	EC50: 3.43 EC50: 8.25	(Hund-Rinke et al., 2018) (Hund-Rinke et al., 2018)
		34	D. magna	EC50: 5.63	(Hund-Rinke et al., 2018)
		30	D. magna	EC50: 1.78	(Liu et al., 2014)
		50	D. magna	EC50: 6.73	(Liu et al., 2014)

Table 3 .

 3 The main characteristic information of these

	NMs come from (European Commission Joint Research Centre, 2014), (European Commission
	Joint Research Centre, 2012), (European Commission Joint Research Centre, 2013),

Table 3 :

 3 Main characteristics of the set of selected NMs

	OECD Designation	Composition	Source	Size (nm)	Specific surface area -BET (m 2 /g)	Crystalline phase	Coating	Dissolution mg/L	ROS production	Image
	NM100	TiO2	Powder	50-90	9.23	Anatase	-	0	14.185
	NM101	TiO2 %TiO2=98.5	Powder	6	316.07	Anatase	-	0	29.674
	NM102	TiO2	Powder	21±10	77.99	Anatase	-	0	20.714
							Polysiloxane;
	NM103	TiO2	Powder	26±10	50.83	Rutile	Al2O3 and	0	10.074
							dimethicone
	NM104	TiO2 %TiO2=90	Powder	26±10	56.26	Rutile (89%)	Al2O3 (6%); (2%) Dimethicone	0	7.938

nm) Specific surface area - BET (m 2 /g)

						Crystalline phase	Coating	Dissolution mg/L	ROS production	Image
	NM105	TiO2 %TiO2=99.5	Powder	21 ± 9	46.17	Anatase (72.6%); Amorphic (9%). Rutile (18.4%);	-	0	27.155
	NM110	ZnO	Powder	151± 55.6	12.4±0.6	Hexagonal	-	21.5	17.312
	NM200	SiO2	Powder	22	189.16	Amorphous	-	38.4	15.883
	NM202	SiO2	Powder	22	204.11	Amorphous	-	16	3.3572
	NM204	SiO2	Powder	21	136.6	Amorphous	-	12.7	3.934

Table 4 :

 4 Preparation volumes of algae, media and NMs intermediate suspension.

Table 5 :

 5 Limits of quantification for the ICP-OES analysis of organisms' samples and water samples.

		Samples of organisms		Samples of water
	Element	LQ of element (µg/L)	LQ of the corresponding metallic oxide (µg/L)	LQ of the sample (g/Kg)	LQ of element µg/L	LQ of the corresponding metallic oxide (µg/L)	LQ of the sample (mg/L)
	Ti	1	1.7	0.028	5	8.3	0.083
	Zn	1	1.2	0.021	5	6.2	0.062
	Si	50	107.1	1.786	10	21.4	0.214
	Ce	2.5	3.1	0.051	2.5	3.1	0.031
	Note:

Table 6 :

 6 Number of R. subcapitata cells in control population for the interaction kinetic tests and the calculation of the coefficient of variation.

	Number of R. subcapitata cells in control population
	04/07/2019 15/07/2019 29/07/2019 19/09/2019
	30693	23395	19212	12061
	32726	25379	16885	12429
	30361	23523	18153	13461
	30556	25683	16338	12234
	31078	23487	16860	12432
	33818	26134	18111	13468
	32150	26921	16697	12574
	33527	23665	17025	12339
	32499	25379	20003	13664
	33688	27055	17510	12043
	31689	23456	18466	11255
	29683	25683	18350	13324
	29673	27269	20177	11770
	29765	23562	17328	12347
	29428	28353	18373	13205
	30011	26940	16937	12235
	29660	23638	18429	12389
		26092	19889	13266
		25025	17185	13135
		26987	18033	11810
		23458	17108	12345
		25973	18362	13179
		25359	19976	12748
		26842	17110	11932
		23503	18473	12332
		25851	17371	13220
			19549	12877
			20038	12025
			17136	12364
			18552	13574
		Coefficient of variation
	5.0%	6.0%	6.3%	4.9%
	mean = 5.6%; standard deviation = 0.7%

10% is chosen as the minimal valid difference

Table 7 :

 7 Percentage difference of the algae-NMs granularity modification between the three tested kinetic times.

						(%f -%i)
	NMs	Concentration mg/L	0.1	0.5	1	2.5	5	7.5	10	12.5	25	50
		From 15min to 1h 1.9	0.4	-0.6 1.7	0.5	4.3	4.5	4.4	5.0	-0.8
	NM100	From 15min to 2h -1.6	2.2* 2.8* 0.7 6.0* 2.4	0.0	5.3* 7.4*	2.3
		From 1h to 2h	-3.5	1.8* 3.4* -1.1 5.5* -1.9 -4.5	0.9	2.4	3.1
		From 15min to 1h -0.6	-0.5	0.0 -0.9 0.1	3.7	4.8	3.6	6.4	6.9
	NM101	From 15min to 2h -0.6	1.2	1.0	1.6	3.8	5.2	7.3	8.7	14.8 27.4
		From 1h to 2h	0.0	1.6	1.0	2.6	3.7	1.5	2.5	5.1	8.4	20.5
		From 15min to 1h 0.3	-0.6	0.3	0.3	0.6	0.8	1.5	0.9	0.6	0.3
	NM102	From 15min to 2h 1.0	0.6	0.9	0.5	2.9	2.8	3.3	3.0	5.3*	4.1
		From 1h to 2h	0.7	1.1	0.6	0.2	2.3	2.0	1.7	2.1	4.7*	3.8
		From 15min to 1h -0.5	0.7	0.6	2.1	0.6 -0.2 -0.4	-0.4	0.0	-0.1
	NM103	From 15min to 2h 0.5	1.3	1.5	3.6	1.1	0.0	-0.4	-0.3	-0.2	-0.5
		From 1h to 2h	1.0	0.6	0.8	1.4	0.4	0.2	0.0	0.1	-0.2	-0.3
		From 15min to 1h 0.4	1.6	0.9	1.3	0.4 -0.1 -0.7	0.4	0.0	0.2
	NM104	From 15min to 2h -0.4	3.3	1.8	0.8	0.0 -0.5	0.4	3.2	3.0	0.5
		From 1h to 2h	-0.8	1.6	0.9 -0.5 -0.4 -0.3	1.1	2.9	3.0*	0.3
		From 15min to 1h 0.9	1.0	2.1	1.6 -0.6 -0.3	1.3	-1.9	-0.2	-0.3
	NM105	From 15min to 2h -0.2	-0.7	-0.6 -0.4 -2.3 -1.5 -0.1	-2.4	-0.5	-0.2
		From 1h to 2h	-1.1	-1.7	-2.7 -2.0 -1.7 -1.2 -1.4	-0.5	-0.3	0.1
		From 15min to 1h 0.0	0.0	0.1	0.0	0.1	0.3	0.0	0.1	0.1	-0.2
	NM200	From 15min to 2h 0.0	0.1	0.4* 0.1	0.3	0.3	0.1	0.1	0.1	-0.2
		From 1h to 2h	0.1	0.1	0.3	0.0	0.2	0.1	0.1	0.1	0.0	0.0
		From 15min to 1h 0.4	0.4	0.2	0.5 -0.2 -0.6	0.1	0.4	0.3	0.4
	NM202	From 15min to 2h 0.3	0.3	0.1	0.6	0.2 -0.4	0.4	0.1	0.1	1.1
		From 1h to 2h	-0.1	-0.1	-0.1 0.1	0.4	0.2	0.3	-0.2	-0.2	0.7
		From 15min to 1h 0.6	0.7	0.0 -0.3 -0.5 0.1	0.5	0.0	-0.4 -1.4*
	NM204	From 15min to 2h 0.7	0.9*	0.4 -0.5 -0.2 0.5	0.2	0.2	0.1	-0.8
		From 1h to 2h	0.1	0.2	0.3 -0.2 0.3	0.4	-0.3	0.3	0.5	0.6
		From 15min to 1h -0.5	-0.1	0.2 2.2* 4.2* -0.5 -0.4	-0.1 -4.0* 7.2
	NM110	From 15min to 2h -0.1	0.2	2.1* 6.0* 6.0* 1.2	1.3	1.6* -3.6* 6.0
		From 1h to 2h	0.4	0.3	2.0* 3.8* 1.8	1.6	1.7* 1.7*	0.4	-1.2
		From 15min to 1h 0.3	1.0	3.2	3.1	3.7	4.3	5.3* 5.9* 7.2*	1.8
	CeO2<25	From 15min to 2h 0.1	0.8	4.4	3.0	3.7 6.5* 13.1* 13.7* 13.5* 8.5
		From 1h to 2h	-0.2	-0.2	1.1 -0.1 0.0	2.2	7.8* 7.8*	6.3	6.7
		From 15min to 1h 0.8	-0.7	0.4	0.6	0.2	0.5	0.0	0.0	0.8	2.3
	CeO2<10	From 15min to 2h 0.3	-0.5	0.9	0.6	1.7	4.9	6.2	2.5	10.0 15.6
		From 1h to 2h	-0.5	0.2	0.6	0.1	1.5	4.4	6.2	2.4	9.1	13.3

Table 8 :

 8 Acute toxicity results EC10, EC25 and EC50 of the OECD 201 test of growth inhibition on freshwater algae. n.a. = Data not available. Data obtained under and belonging to the NANoREG2 project.

	NMs	Acute toxicity results (mg/L) EC10 EC25 EC50
	TiO2-NM100	8.498	9.867	11.457
	TiO2-NM101	0.608	1.686	4.675
	TiO2-NM102	1.091	2.614	6.266
	TiO2-NM103	1.441	6.099	25.816
	TiO2-NM104	2.140	8.615	34.675
	TiO2-NM105	0.930	2.212	5.263
	ZnO-NM110	0.079	0.101	0.129
	SiO2-NM200	5.649	7.896	11.038
	SiO2-NM202	2.324	3.816	6.265
	SiO2-NM204	3.568	8.730	21.359
	CeO2>10nm	0.740	n.a.	9.150
	CeO2>25nm	3.878	n.a.	27.455

Table 1 .

 1 List of housekeeping and target genes used for the RT-qPCR analysis. Gene Bank accession numbers and primer sequences are provided.

	Number	Gene	Description	Function	Gene bank accession	Forward primer (50-30)	Reverse primer (50-30)	Reference
	I	β-actin	Actin	Housekeeping gene (structure)	AJ292554	GCCCTCTTCCAGCCCTCATTCT TGGGGCAAGGGCGGTGATTT	(Maselli et al., 2017)
	II	18s	18S ribosomal RNA	Housekeeping gene (protein biosynthesis)	AF070104	CGCTCTGAATCAAGGGTGTT	TGTCCGACCGTGAAGAGAGT	(Heckmann et al., 2006)
	III	gapdh	Glyceraldehyde-3-phosphate dehydrogenase	Housekeeping gene (Glycolysis)	AJ292555	GGCAAGCTAGTTGTCAATGG	TATTCAGCTCCAGCAGTTCC	(Heckmann et al., 2006)
	IV	ubc	Ubiquitin Conjugating Enzyme	Housekeeping gene		TCACCTGCACTCACCATTTC	AATCTCCGGAACCAAAGGAT	(Heckmann et al., 2006)
	1	sod	Superoxide dismutase	Destroy free superoxide radicals	DW985490	CTTGACTGGGCTATTGA	GTTATTGCTGTCGGATGT	(Chen et al., 2017)
	2	gst	Glutathione-S-transferase	Detoxify xenobiotics	DV437830	TCAGGCTGGTGTTGAGTTTG	GAGCAAGCATTTGTCCATCA	(Poynton et al., 2012) (Seyoum et al., 2021)
	3	cyp	Cytochrome P450-314 family	Impaired detoxification of xenobiotics	AB257771	TCTTGGGTCGGCGTCTGGGA	TCGCGGGTGTCAACGCCTTC	(Seyoum et al., 2021)
	4	hsp70	Heat shock protein 70	ATP binding	EU514494	CCTTAGTCATGGCTCGTTCTC	TCAAGCGGAACACCACTATC	(Dominguez et al., 2015)
								(Poynton et al., 2007)
	5	mt	Metallothionein	Metal binding and detoxification	DV437826	TTGCCAAAACAATTGCTCAT	CACCTCCAGTGGCACAAAT	(Poynton et al., 2008) (Dominguez et al.,
								2015)
	6	rev1	DNA repair gene	DNA damage	DM03449	ATGCGTTAGGCGTCAATACC	TACAAGGTTTGCCCTTGCTT	(Poynton et al., 2012)
	7	flot	Flotillin	Phagocytosis	DM00331	GCGACCAATTCGCTTCTCTA	TGGTGAAGGAAAGGATTTCG	(Poynton et al., 2012)
	8	jhe	Juvenile Hormone Esterase	Development and molting	DW724473	GAAGCCACTCGCATGGAAGC	CCGGTGAAAGCGCAGGCAGT	(Seyoum et al., 2021)
	9	ecr	Ecdysone receptor A	Molting and metamorphose	AB274823 CAGGCACATCAACATCAACAA	GGCGACATGGAATCGACA	(Seyoum et al., 2021)
	10	vtg	Vitellogenin	Reproduction	AB252738	TTAAGGCTCGCCGCATCGGA	ACCTCGGCGCAAAGCACGAA	(Maselli et al., 2017)
	11	amy	α-amylase	Digestive enzyme activity	DV437794	CGGTGTCTCCTATGGTGGAT	TCCCGTCAGAAGAAGGACAT	(Poynton et al., 2007)

Table 2 .

 2 Physico-chemical characteristics of the tested NMs.

		Pristine	Specific			Hydrodynamic	Z potential
	NMs	NMs size	surface area	Crystalline phase	Coating	diameter in ISO	in ISO
		nm	-BET m 2 /g			media d. nm	media mV
	TiO2-NM100	50-90	9.23	Anatase	-	361.3 ± 14.7	-7.6 ± 0.87
	TiO2-NM101	6	316.07	Anatase	-	792.2 ± 46.6	-8.4 ± 0.78
	TiO2-NM102	21±10	77.99	Anatase	-	2279.3 ± 295.4 a -5.5 ± 0.71
					Polysiloxane;
	TiO2-NM103	26±10	50.83	Rutile	Al2O3 and	889.7 ± 23.3	3.9 ± 0.02
					dimethicone
					Al2O3 (6%);
	TiO2-NM104	26±10	56.26	Rutile (89%)	Dimethicone	677.7 ± 97.1	4.7 ± 0.02
					(2%)
				Anatase (72.6%);
	TiO2-NM105	21 ± 9	46.17	Rutile (18.4%);	-	844.4 ± 124.7	-1.9 ± 0.46
				Amorphic (9%).
	ZnO-NM110 151± 55.6	12.4	Hexagonal		1144.5 ± 153.0 a -2.1 ± 0.29
	SiO2-NM200	22	189.16	Amorphous	-	1027.1 ± 307.8 a -14.9 ± 1.10
	SiO2-NM202	22	204.11	Amorphous	-	684.5 ± 5.6	-16.4 ± 0.79
	SiO2-NM204	21	136.6	Amorphous	-	987.0 ± 79.6 a -16.9 ± 0.53
					Tri-
	CeO2>10nm	<10	84.15	Cubic	ammonium	361.3 ± 107.8	-13.3 ± 0.55
					citrate
	CeO2>25nm	<25	33.66	Cubic	-	792.2 ± 42.8	-7.0 ± 1.15

a

To consider with care as size cannot be accurately determined by DLS above 1000 nm.

Table 3 .

 3 Acute toxicity results EC10, EC25 and EC50 of the OECD 202 test of mobility inhibition on D. magna.

	NMs	Acute toxicity results (mg/L) EC10 EC25 EC50
	TiO2-NM100	>100	>100	>100
	TiO2-NM101	14.8	24.1	39.1
	TiO2-NM102	>100	>100	>100
	TiO2-NM103	>100	>100	>100
	TiO2-NM104	>100	>100	>100
	TiO2-NM105	>100	>100	>100
	ZnO-NM110	9.43	14.58	22.48
	SiO2-NM200	>100	>100	>100
	SiO2-NM202	>100	>100	>100
	SiO2-NM204	>100	>100	>100
	CeO2>10nm	n.a.	n.a.	>50
	CeO2>25nm	>100	>100	>100

Table 4 .

 4 Acute toxicity results EC10, EC25 and EC50 of the OECD 202 test of mobility inhibition on D. magna. Not available. Limits of quantification are: 1µg/L for Ti; 1µg/L for Zn; 50 µg/L for Si; and 2.5 µg/L for Ce.

	NMs	Body burden (g/kg dry weight) Uptake 24h Elimination 5d
	TiO2-NM100	36.42 ± 4.53	0.30 ± 0.06
	TiO2-NM101	44.41 ± 5.34	0.11 ± 0.16
	TiO2-NM102	76.49 ± 4.33	0.54 ± 0.29
	TiO2-NM103	114.23 ± 22.98	0.38 ± 0.06
	TiO2-NM104	41.82 ± 7.98	0.62 ± 0.07
	TiO2-NM105	57.76 ± 5.99	1.82 ± 0.87
	ZnO-NM110	0.67 ± 0.05	0.10 ± 0.01
	SiO2-NM200	47.55 ± 10.60	<lq
	SiO2-NM202	<lq	<lq
	SiO2-NM204	24.00 ± 3.18	2.01 ± 1.44
	CeO2>10nm	78.43 ± 8.77	0.38 ± 0.38
	CeO2>25nm	47.74 ± 8.41	0.56 ± 0.46
	n.a.=

Metal dosing of D. magna body burden (g/Kg) after 24h of exposure and after 5 days of elimination to 12 different NMs. The metal concentrations of some samples are under the limit of quantification (<lq) of the ICP-OES.

Table 14 :

 14 General data matrix used for the principal component analyses for NMs clustering Part I.

		Prist size	SSA	Dissol	ROS	Size stock	Size algae	Size ISO	Zpot stock	Zpot algae	Zpot ISO	Int Raphi	Int Desmo	Int Chlo	Up 24h	Elim 120h
	NM100	1.845	0.963	-0.671	1.152	2.365	2.696	2.558	0.111	-0.782 -0.060	0.353	-0.260	0.067	-0.344 -0.294
	NM101	0.778	2.499	-0.672	1.472	2.652	2.846	2.898	-0.034 -0.330 -0.168	0.119	0.372	0.502	-0.094 -0.816
	NM102	1.322	1.892	-0.673	1.316	3.678	3.363	3.355	-0.026 -0.219	0.213	0.400	0.461	0.616	0.917	0.099
	NM103	1.415	1.706	-0.674	1.003	2.225	3.381	2.949 0.6714 2.441	1.500	0.728	1.470	0.876	2.055	-0.129
	NM104	1.415	1.750	-0.675	0.899	2.245	3.380	2.828	0.769	1.665	1.600	1.177	1.193	1.054	-0.186	0.371
	NM105	1.322	1.665	-0.676	1.433	3.285	3.587	2.923	0.003	-0.223	0.705	1.158	0.714	1.088	0.325	2.621
	NM110	2.179	1.093	0.957	1.238	2.403	3.494	3.392	1.190	0.034	0.682	0.701	0.043 0.4261 -1.461 -0.702
	NM200	1.342	2.276	2.237	1.201	2.379	2.425	3.049	-1.203 -0.495 -1.051	-1.30	-1.296 -1.318 -0.017	0.979
	NM202	1.342	2.309	0.541	0.526	2.239	2.396	2.250	-1.200 -0.699 -1.259 -1.290 -1.293 -1.322 -1.432 -0.908
	NM204	1.322	2.135	0.291	0.595	2.335	2.392	3.010	-1.116 -0.647 -1.327	-1.26	-1.116 -1.311 -0.733 -0.908
	CeO2<10nm 1.000	1.924	0.000	1.084	0.944	1.404	2.831	-1.004 -0.463 -0.843 -1.312 -1.104 -1.256	0.971	-0.335
	CeO2<25nm 1.398	1.527	0.000	1.085	2.281	3.632	2.994	1.839	-0.281	0.011	0.529	0.818	0.578	0.001	0.023

Acknowledgments

The last three years have been a wonderful journey of learning. I would like to begin thanking my thesis supervisor Prof. Catherine Mouneyrac for her priceless advice, for the academic guidance all along and for the positive feedback that I have always received. Next, I thank my co-supervisor Dr. Amélie Châtel for her willingness to get involved in this project, for all the constructive comments on my work, for receiving me at the Catholic University of the West and for the support she gave me during my stay.

https://tmblr-Interviewed about my research subject for the "Yakademia Podcast". This scientific podcast is https://open.spotify

Supplementary results

Modeled profiles of uptake and elimination of NMs

The modeled profiles of the uptake and elimination of NMs by D. magna, along with the measured points, are presented in Figure 33. Median is represented by orange plain line and the uncertainty band by the gray zone, delimited by the 2.5% and 97.5% quantiles in orange dotted lines. In the previous chapter, the uptake and elimination behavior of the selected set of NMs was determined and important differences were found. But the relationship between the different uptake and eliminations profiles of NMs and their effects on the organisms was not yet determined.

The emergence of ecotoxicogenomic analytical tools for HTS of biomolecules have however revolutionized the way in which toxicologists explore the impact of chemicals or other stressors on biota [START_REF] Oberholster | Applying genotoxicology tools to identify environmental stressors in support of river management[END_REF]. Transcriptomics has been put forward as a relevant HST method for analyzing the genes expression in response to toxicants and focusing on characterizing sublethal mechanisms of toxicity. This can potentially provide a way to screen NMs in a time-efficient and cost-effective manner [START_REF] Barrick | The role of high-throughput screening in ecotoxicology and engineered nanomaterials[END_REF].

Among the many advantages that transcriptomics have, the possibility of study different process at cellular and individual level is a great benefit. Since NMs are known to affect organisms not only at the biochemical level but also having some physical interactions (i.e. with the carapace of the swimming behavior), it is important to study all the spheres of NMs impact.

Samples from the uptake and elimination behavior experiment were took to be analyzed by RT-qPCR with the goal of answering the following questions:

• How are the organisms' processes affected by the exposure to NMs? And how the cellular and individual mechanisms are impacted with the uptake of NMs?

• How the exposed organisms recover during the elimination process from NMs?

And how the cellular and individual mechanisms respond?

• Are the differences in the genetic expressions of the organisms exposed to NMs correlated to their uptake and elimination behaviors? or to their acute toxicity?

• It is possible to identify if some of the NMs characteristics could be related to the toxicity responses observed?

• How this analysis can be relevant for regulatory purposes?

Chapter 6: The NMs characteristics and nano-bio interaction as elements for NMs grouping

Introduction and context

Within the production and release of the increasingly great diversity of NMs;

international regulatory bodies, governments, industrials and scientists have started to research for grouping and read-across strategies for NMs. But the main challenge that these projects have faced is the limited availability of data due to the non-harmonized experimental conditions. Systematic studies as the one presented in this thesis allow to generate a lot of information for each one of the studied NMs. All these comparable data obtained under the same conditions for every NM allow to better understand the nano-bio interactions for risk assessment purposes in a global context of need of these types of analyses.

In the aim of classifying NMs based on their interaction with biological organisms, a statistical analysis of the correlations of the entire known parameters may help to find NMs that interact in a similar way or not with the organisms and the possible descriptors of those similarities. In this context, and based on the obtained and given information of this set of NMs, the questions that this chapter tries to answer are as follows:

• How are the studied NMs similar or different based on their physicochemical (intrinsic and extrinsic or media dependent) characteristics?

• How are the studied NMs similar or different based on the interrelation of their physicochemical characteristics and the nano-bio interactions as well as toxicity towards the different studied organisms?

• How are the studied NMs similar or different based on their early molecular effects determined by transcriptomic analysis?

• Which is the relevance of the nano-bio interaction and early molecular effects for NMs grouping strategies?

Annex 1: Media compositions To prepare 1L of LC media, add the indicated volume of concentrated solutions to 1L of MQ water in a clean glass container. Once the media has be distributed in the Erlenmeyer flasks of about 100 mL per flask, they can be closed using aluminum paper and they must be sterilized. To prepare 1L of Algae OECD growth media, add the indicated volume of concentrated solutions to 1L of sterile water in sterilized glass container. The Erlenmeyer flask used to distribute the Algae OECD growth media in 100 mL portions, must also be sterilized.

Annex 3: Supplementary modelling results

All the parameters presented below account for the accuracy of the modelling results.

For Table 13 andFigures 43 to 46: data and descriptions of the modeling parameters are obtained from the modeling reports generated by : http://lbbe-shiny.univ-lyon1.fr/mosaicbioacc/ and using the downable R script to obtain better results, adding a 4th MCMC chain and more sampling (niter = 300000 and thin = 80). Part II.

BCF