Alice Pradel
email: alice.pradel@univ-rennes1.fr

Séléna Ferreres

Cloé Veclin

El Hadri

Maud Gautier

Bruno Grassl

Julien Gigault
email: julien.gigault@takuvik.ulaval.ca

Stabilization of fragmental polystyrene nanoplastic by natural organic matter: Insight into mechanisms

Keywords: Plastic debris, Environmental fate, Aggregation, Ionic strength, Morphology

The increasing amount of plastic debris in the environment and its disintegration into submicrometric particles is a cause for concern. Due to the colloidal nature of nanoplastics, their environmental fate should be investigated separately from that of microplastics. Abiotic factors greatly influence nanoplastics' stability. This will affect its residence time in the hydrosphere. So, we investigated the behavior of two different nanoplastic models (with different sizes and shapes) regarding ionic strength, pH, and varying concentrations of two natural organic matters: humic acid and sodium alginate. The results demonstrate that both natural organic matters enhanced the aqueous stability of nanoplastics over time at high ionic strengths. Depending on the organic matter's nature, different stabilizing mechanisms were revealed using dynamic light scattering and asymmetrical flow field flow fractionation coupled to static light scattering. Humic acid provides electrostatic repulsion between particles, and some larger humic acid molecules provide a steric hindrance. Sodium alginate sorbs onto and bridges separate particles and small aggregates of nanoplastics. The covered particles are stabilized by steric hindrance. The results highlight the importance of considering natural organic matters' properties when assessing nanoplastics behavior in the environment.

INTRODUCTION

As the use of plastic-based materials increases, plastic waste in the environment increases proportionally [START_REF] Geyer | Use, and Fate of All Plastics Ever Made[END_REF][START_REF] Jambeck | Plastic Waste Inputs from Land into the Ocean[END_REF] . These last five years, it was demonstrated that plastic debris could persist as nanoplastic (< 1µm) before eventual mineralization of the polymer [START_REF] Gigault | Marine Plastic Litter: The Unanalyzed Nano-Fraction[END_REF][START_REF] Lambert | Characterisation of Nanoplastics during the Degradation of Polystyrene[END_REF][START_REF] Zhu | Photochemical Dissolution of Buoyant Microplastics to Dissolved Organic Carbon: Rates and Microbial Impacts[END_REF] . Environmental sampling of plastic debris at the ocean surface, coupled to numerical modeling, suggests that a substantial part of all the plastic debris is composed of nanoplastics [START_REF] Koelmans | All Is Not Lost: Deriving a Top-down Mass Budget of Plastic at Sea[END_REF][START_REF] Ter Halle | Nanoplastic in the North Atlantic Subtropical Gyre[END_REF][START_REF] Van Sebille | A Global Inventory of Small Floating Plastic Debris[END_REF] . Since nanoplastics are an emerging contaminant, their environmental fate should be better described. While airborne transport of microplastics is increasingly coming under scrutiny [START_REF] Allen | Atmospheric Transport and Deposition of Microplastics in a Remote Mountain Catchment[END_REF][START_REF] Evangeliou | Atmospheric Transport Is a Major Pathway of Microplastics to Remote Regions[END_REF] , water remains the environmental compartment where most plastic debris is found [START_REF] Gesamp | Sources, Fate and Effects of Microplastics in the Marine Environment: Part Two of a Global Assessment[END_REF][START_REF] Horton | Microplastics in Freshwater and Terrestrial Environments: Evaluating the Current Understanding to Identify the Knowledge Gaps and Future Research Priorities[END_REF] and where oxidative and hydrolytic conditions are favorable to plastic degradation [START_REF] Chamas | Degradation Rates of Plastics in the Environment[END_REF][START_REF] Gewert | Pathways for Degradation of Plastic Polymers Floating in the Marine Environment[END_REF][START_REF] Julienne | From Macroplastics to Microplastics: Role of Water in the Fragmentation of Polyethylene[END_REF][START_REF] Min | Ranking Environmental Degradation Trends of Plastic Marine Debris Based on Physical Properties and Molecular Structure[END_REF] . As such, it is crucial to understand the behavior of nanoplastics in aqueous systems.

To describe a colloidal material's environmental fate in aqueous systems, successive and complementary approaches consist of modeling simple environmental systems in the lab, using these results to establish numerical simulations, and, finally, confronting these simulations with the analysis of environmental samples [START_REF] Buffle | A Generalized Description of Aquatic Colloidal Interactions: The Three-Colloidal Component Approach[END_REF] . Based on this approach, experimental systems describing nanoplastics' fate have emerged [START_REF] Alimi | Microplastics and Nanoplastics in Aquatic Environments: Aggregation, Deposition, and Enhanced Contaminant Transport[END_REF] , especially concerning porous media [START_REF] Dong | Role of Surface Functionalities of Nanoplastics on Their Transport in Seawater-Saturated Sea Sand[END_REF][START_REF] Keller | Transport of Nano-and Microplastic through Unsaturated Porous Media from Sewage Sludge Application[END_REF][START_REF] Liu | Aging Significantly Affects Mobility and Contaminant-Mobilizing Ability of Nanoplastics in Saturated Loamy Sand[END_REF] and water [START_REF] Li | Interactions between Nano/Micro Plastics and Suspended Sediment in Water: Implications on Aggregation and Settling[END_REF][START_REF] Oriekhova | Heteroaggregation of Nanoplastic Particles in the Presence of Inorganic Colloids and Natural Organic Matter[END_REF] . Nanoplastics' stability in water is generally determined by measuring changes in their size and sedimentation rates. Using this approach, the stability of nanoplastics has been assessed in natural waters [START_REF] Oriekhova | Heteroaggregation of Nanoplastic Particles in the Presence of Inorganic Colloids and Natural Organic Matter[END_REF][START_REF] Bergami | Polystyrene Nanoparticles Affect the Innate Immune System of the Antarctic Sea Urchin Sterechinus Neumayeri[END_REF][START_REF] Chen | Sedimentation of Nanoplastics from Water with Ca/Al Dual Flocculants: Characterization, Interface Reaction, Effects of PH and Ion Ratios[END_REF][START_REF] Della Torre | Accumulation and Embryotoxicity of Polystyrene Nanoparticles at Early Stage of Development of Sea Urchin Embryos Paracentrotus Lividus[END_REF][START_REF] González-Fernández | Do Transparent Exopolymeric Particles (TEP) Affect the Toxicity of Nanoplastics on Chaetoceros Neogracile?[END_REF][START_REF] Manfra | Comparative Ecotoxicity of Polystyrene Nanoparticles in Natural Seawater and Reconstituted Seawater Using the Rotifer Brachionus Plicatilis[END_REF][START_REF] Ramirez | Behavior of TiO2 and CeO2 Nanoparticles and Polystyrene Nanoplastics in Bottled Mineral, Drinking and Lake Geneva Waters. Impact of Water Hardness and Natural Organic Matter on Nanoparticle Surface Properties and Aggregation[END_REF][START_REF] Seoane | Polystyrene Microbeads Modulate the Energy Metabolism of the Marine Diatom Chaetoceros Neogracile[END_REF][START_REF] Singh | Understanding the Stability of Nanoplastics in Aqueous Environments: Effect of Ionic Strength, Temperature, Dissolved Organic Matter, Clay, and Heavy Metals[END_REF][START_REF] Song | Fate and Transport of Nanoplastics in Complex Natural Aquifer Media: Effect of Particle Size and Surface Functionalization[END_REF][START_REF] Tallec | Surface Functionalization Determines Behavior of Nanoplastic Solutions in Model Aquatic Environments[END_REF] , in deionized water with various ionic compositions, ionic strengths, and pHs [START_REF] Liu | Aging Significantly Affects Mobility and Contaminant-Mobilizing Ability of Nanoplastics in Saturated Loamy Sand[END_REF][START_REF] Li | Interactions between Nano/Micro Plastics and Suspended Sediment in Water: Implications on Aggregation and Settling[END_REF][START_REF] Chen | Sedimentation of Nanoplastics from Water with Ca/Al Dual Flocculants: Characterization, Interface Reaction, Effects of PH and Ion Ratios[END_REF][START_REF] Singh | Understanding the Stability of Nanoplastics in Aqueous Environments: Effect of Ionic Strength, Temperature, Dissolved Organic Matter, Clay, and Heavy Metals[END_REF][START_REF] Cai | Effects of Inorganic Ions and Natural Organic Matter on the Aggregation of Nanoplastics[END_REF][START_REF] Dong | Cotransport of Nanoplastics (NPs) with Fullerene (C60) in Saturated Sand: Effect of NPs/C60 Ratio and Seawater Salinity[END_REF][START_REF] Magrì | Laser Ablation as a Versatile Tool To Mimic Polyethylene Terephthalate Nanoplastic Pollutants: Characterization and Toxicology Assessment[END_REF][START_REF] Mao | Nanoplastics Display Strong Stability in Aqueous Environments: Insights from Aggregation Behaviour and Theoretical Calculations[END_REF][START_REF] Wu | Effect of Salinity and Humic Acid on the Aggregation and Toxicity of Polystyrene Nanoplastics with Different Functional Groups and Charges[END_REF][START_REF] Yu | Aggregation Kinetics of Different Surface-Modified Polystyrene Nanoparticles in Monovalent and Divalent Electrolytes[END_REF][START_REF] Zhang | Aquatic Behavior and Toxicity of Polystyrene Nanoplastic Particles with Different Functional Groups: Complex Roles of PH, Dissolved Organic Carbon and Divalent Cations[END_REF] , in the presence of NOM and suspended sediments [START_REF] Li | Interactions between Nano/Micro Plastics and Suspended Sediment in Water: Implications on Aggregation and Settling[END_REF][START_REF] Oriekhova | Heteroaggregation of Nanoplastic Particles in the Presence of Inorganic Colloids and Natural Organic Matter[END_REF][START_REF] González-Fernández | Do Transparent Exopolymeric Particles (TEP) Affect the Toxicity of Nanoplastics on Chaetoceros Neogracile?[END_REF][START_REF] Singh | Understanding the Stability of Nanoplastics in Aqueous Environments: Effect of Ionic Strength, Temperature, Dissolved Organic Matter, Clay, and Heavy Metals[END_REF][START_REF] Tallec | Surface Functionalization Determines Behavior of Nanoplastic Solutions in Model Aquatic Environments[END_REF][START_REF] Cai | Effects of Inorganic Ions and Natural Organic Matter on the Aggregation of Nanoplastics[END_REF][START_REF] Magrì | Laser Ablation as a Versatile Tool To Mimic Polyethylene Terephthalate Nanoplastic Pollutants: Characterization and Toxicology Assessment[END_REF][START_REF] Wu | Effect of Salinity and Humic Acid on the Aggregation and Toxicity of Polystyrene Nanoplastics with Different Functional Groups and Charges[END_REF][START_REF] Yu | Aggregation Kinetics of Different Surface-Modified Polystyrene Nanoparticles in Monovalent and Divalent Electrolytes[END_REF][START_REF] Zhang | Aquatic Behavior and Toxicity of Polystyrene Nanoplastic Particles with Different Functional Groups: Complex Roles of PH, Dissolved Organic Carbon and Divalent Cations[END_REF][START_REF] Saavedra | Influence of Nanoplastic Surface Charge on Eco-Corona Formation, Aggregation and Toxicity to Freshwater Zooplankton[END_REF] and in the presence of extracellular polymeric substances (EPS) [START_REF] Mao | Nanoplastics Display Strong Stability in Aqueous Environments: Insights from Aggregation Behaviour and Theoretical Calculations[END_REF][START_REF] Summers | Agglomeration of Nano-and Microplastic Particles in Seawater by Autochthonous and de Novo-Produced Sources of Exopolymeric Substances[END_REF] . Most studies have used polystyrene (PS) latex spheres, which are perfectly smooth, spherical, and monodisperse in size. Recently, the stability of more environmentally relevant models, such as aged polystyrene (PS) latex spheres, laser-ablated PS, or fragmental PET, has been studied [START_REF] Mao | Nanoplastics Display Strong Stability in Aqueous Environments: Insights from Aggregation Behaviour and Theoretical Calculations[END_REF][START_REF] Yu | Aggregation Kinetics of Different Surface-Modified Polystyrene Nanoparticles in Monovalent and Divalent Electrolytes[END_REF][START_REF] Dong | Aggregation Kinetics of Fragmental PET Nanoplastics in Aqueous Environment: Complex Roles of Electrolytes, PH and Humic Acid[END_REF][START_REF] Liu | Aggregation Kinetics of UV Irradiated Nanoplastics in Aquatic Environments[END_REF] .

While these studies allow the emergence of global trends concerning nanoplastic stability, they also have inconsistent conclusions. For example, nanoplastic models have been observed to be both stable and unstable in artificial seawater [START_REF] Tallec | Surface Functionalization Determines Behavior of Nanoplastic Solutions in Model Aquatic Environments[END_REF][START_REF] Okshevsky | Biofilm Formation by Marine Bacteria Is Impacted by Concentration and Surface Functionalization of Polystyrene Nanoparticles in a Species-specific Manner[END_REF] , and in the presence of iron [START_REF] Oriekhova | Heteroaggregation of Nanoplastic Particles in the Presence of Inorganic Colloids and Natural Organic Matter[END_REF][START_REF] Cai | Effects of Inorganic Ions and Natural Organic Matter on the Aggregation of Nanoplastics[END_REF] . Such discrepancies can be explained first by the physical and chemical properties of the nanoplastics models used (size, shape, surface functionalization, composition, purity, etc.), which are known to strongly affect the behavior of colloidal materials. Additionally, nanoplastics are strongly sensitive to the media's properties (type, concentration, and speciation of electrolytes, nature of the organic matter, pH). Indeed, according to the relative concentration of spherical PS nanoplastic models, NOM, and cations, opposite behaviors have been observed [START_REF] Oriekhova | Heteroaggregation of Nanoplastic Particles in the Presence of Inorganic Colloids and Natural Organic Matter[END_REF][START_REF] Yu | Aggregation Kinetics of Different Surface-Modified Polystyrene Nanoparticles in Monovalent and Divalent Electrolytes[END_REF] .

In light of these observations, the stability of two nanoplastic models was studied. The first model is a monodisperse polystyrene latex (PSL) sphere. The second model is produced from the mechanically degraded primary microplastic (PS pellets) and, as such, is more environmentally relevant due to its irregular, asymmetrical shape and polydisperse size. NPs' aqueous stability was assessed at different ionic strengths (5 to 770 mmol L -1 NaCl) and in the presence of varying concentrations (0.005 to 140 mg L -1 ) of two NOMs which have different properties: humic acid (HA) and sodium alginate (SA).

HA has a relatively compact structure and amphiphilic properties, whereas SA has a more linear structure with hydrophilic properties. HA represents terrestrial organic matter, whereas SA represents marine organic matter and is a significant component of EPS produced by microbial communities [START_REF] Flemming | The Biofilm Matrix[END_REF][START_REF] Stumm | Aquatic Chemistry : Chemical Equilibria and Rates in Natural Waters[END_REF] .

It was demonstrated that both NOMs stabilize the environmentally relevant NP model at high ionic strength. However, due to their different physico-chemical properties, the NOMs have different stabilizing mechanisms. These were characterized by asymmetrical flow field flow fractionation coupled to static light scattering (A4F-SLS) and confirmed by dynamic dynamic light scattering (DLS).

The present work discusses these mechanisms and their possible implication for the fate of nanoplastics in both terrestrial and marine environments.

EXPERIMENTAL

Sample preparation

All aqueous solutions and dispersions were prepared with analytical grade deionized (DI) water (Millipore, 18.2 M). A stock solution of NaCl (solid, LabKem ExtraPure) at 1.80 mol L -1 was prepared.

The pH of all solutions was fixed at pH of 5, 6.5, or 8 using NaOH (Fisher Scientific, Analytical Grade) and HCl (70%, Sigma Aldrich, ACS Grade). All solutions and dispersions were stored at 4 o C in the dark before use. Two nanoplastic (NPs) models were used in this study and are described in Table 1 and illustrated in Figure S1. Carboxylated polystyrene latex spheres of 200 nm (PSL COOH) are purchased from Polysciences© (Polybead® Carboxylate Orange Dyed Microspheres 0.20 μm, Warrington USA).

A stock dispersion at a concentration of 100 mg L -1 was prepared. A NP model with irregular and polymorphic shapes (NPT-P) was produced by the mechanical abrasion of industrial-grade polystyrene (PS) pellets (Total, Paris, France) as described by El Hadri et al. (2020) [START_REF] El Hadri | Nanoplastic from Mechanically Degraded Primary and Secondary Microplastics for Environmental Assessments[END_REF] . The pellets are composed of primary (-P) PS, which contains no additives and has not been aged. Due to the less stable nature of the NPT-P compared to PSL COOH, the experiments presented here used different batches of NPT-P to avoid a bias brought about by the aging of the stock dispersion. Each batch was produced using PS from the same degradation round. Before each experiment, the size of the nanoplastic dispersions was verified with DLS measurements. Concentration of the stock NPT-P solution was measured with a Total Carbon Analyzer (Shimadzu TOC-V CSH) and varied between 22 and 35 mg L -1 . According to the molecular composition of PS, 1 mg L -1 of organic carbon was converted to 1.08 mg L -1 NPT-P.

Table 1: Characteristics of the two nanoplastic (NP) models. The polydispersity index (PDI) is defined as the variance of the

Gaussian-fitted size distribution. The aspect ratio is defined as the ratio of the length of the major axi and minor axi as determined by TEM images (Figures S1 andS2). ) under continual agitation with a magnetic stirrer. Then, the solution was mixed at 350 rpm for 24h. pH was then fixed to either 5, 6.5, or 8 using 0.1 mol L -1 HCl. The concentrations of the NOM stock solutions were determined with a Total Carbon Analyzer (Shimadzu TOC-V CSH). According to the NOM's molecular composition, 1 mg L -1 of organic carbon was converted to 1.6 mg L -1 HA and 2.8 mg L -1 SA.

Size characterization

Hydrodynamic diameters (dH) were determined by dynamic light scattering (DLS) probe (see Supplemental Information, S1).

Kinetics of Colloidal Aggregation

The kinetics of nanoplastic aggregation were determined by measuring the z-average hydrodynamic diameters (dzH) of the dispersions over one hour. A total volume of 3 mL was prepared by adding NaCl and DI water to the vial, followed by NOM (when it was studied), and vigorously mixing the solution. Finally, the nanoplastic dispersion was added to the vial, marking the beginning of the kinetic study. All kinetic studies were performed in triplicate. The aggregation rate (k) was determined from the slope of the one-hour-long kinetic study, according to equation ( 1):

( 𝑑𝑑 𝑧𝐻 (𝑡) 𝑑𝑡 ) 𝑡→ 0 ∝ 𝑘𝑁 0 (1)
where dzH(t) is the hydrodynamic diameter of aggregates as a function of time t and N0 is the initial number-based particle concentration. Statistical analyses were operated using one-way ANOVA.

Pairwise comparisons of aggregation rates were made using Tukey's method.

At low ionic strengths, electrostatic repulsion between particles is high due to a thick electrical double layer (EDL): the colloidal dispersion is said to be in the reaction-limited aggregation (RLA) regime. As ionic strength increases, electrostatic repulsion decreases, and the aggregation rate increases. At an ionic strength corresponding to the critical coagulation concentration (CCC), the interparticle energy barrier is eliminated, aggregation rate is maximal (kfast), and the diffusion-limited aggregation (DLA) regime is reached. The attachment efficiency α describes aggregation kinetics by normalizing aggregation rates under RLA regime (k) by the DLA regime (kfast):

𝛼 = 𝑘 𝑘 fast = ( 𝑑𝐷 𝑧𝐻 (𝑡) 𝑑𝑡 ) 𝑡→0 ( 𝑑𝐷 𝑧𝐻 (𝑡) 𝑑𝑡 ) 𝑡→0,fast (2)

Derjaguin Landau Verwey Overbeek (XDLVO) theory of colloidal stability

The total interaction energy as a function of the distance separating the particles, G tot (h), is calculated as the sum of the Lifshitz-van der Waals attraction, G LW (h), the electrical double layer (EDL) repulsion, G EDL (h), and the Lewis acid-base energy of interaction G AB (h). The surface interaction energy was calculated at an ionic strength of 5 mmol L -1 . Particle diameters and zeta-potential are presented in Table 1.

The Lifshitz-van der Waals component, G LW (h), was calculated using the expression of the retarded van der Waals interactions between two identical approaching spheres proposed by Gregory (1981) [START_REF] Gregory | Approximate Expressions for Retarded van Der Waals Interaction[END_REF] :

𝐺 𝐿𝑊 = - 𝐻𝑟 𝑝1 𝑟 𝑝2 6(𝑟 𝑝1 +𝑟 𝑝2 )ℎ [1 - 𝑏ℎ 𝜆 𝑙𝑛 (1 + 𝜆 𝑏ℎ )] (3)
where H is the Hamaker constant of polystyrene particles interacting through water, equal to 1. 23 10 -20 J; rp1 and rp2 are the radii of particles 1 and 2, respectively; b is an empirically defined constant, b = 5.32;

and λ is the characteristic wavelength of the interaction with a value of ~100 nm [START_REF]Particle Deposition and Aggregation: Measurement, Modelling and Simulation[END_REF] .

The electrical double layer repulsion G EDL (h) was calculated using the expression proposed by Hogg et al. (1966) [START_REF] Hogg | Mutual Coagulation of Colloidal Dispersions[END_REF] :

𝐺 𝐸𝐷𝐿 = 𝜋𝜀 𝑟 𝑝1 𝑟 𝑝2 (𝑟 𝑝1 +𝑟 𝑝2 ) [2𝜉 𝑝1 𝜉 𝑝2 𝑙𝑛 ( 1+𝑒 -𝜅ℎ 1-𝑒 -𝜅ℎ ) + (𝜉 𝑝1 2 + 𝜉 𝑝2 2 )ln (1 -𝑒 -2𝜅ℎ )] ( 4
)
where ε is the permittivity of the medium, equal to 6.95 10 -10 C 2 .J -1 .m -1 , ζp1, and ζp2 are the surface charges of particles 1 and 2, respectively, approximated by the zeta potential; and κ is the inverse of the EDL thickness (Debye Huckel length reciprocal length), determined by the following equation:

𝜅 = [ 𝑒 2 𝜀𝑘 𝐵 𝑇 ∑𝑖𝑧 𝑖 𝑛 𝑖 ] 2 (5)
where e is the charge of the electron; kB the Boltzmann constant, T the temperature, zi the valency of the ions i, and ni the number of ions i per unit volume.

The Lewis acid-base energy of interaction G AB (h) of our system is the expression proposed by van Oss (1993) [START_REF] Van Oss | Acid-Base Interfacial Interactions in Aqueous Media[END_REF] :

𝐺 𝐴𝐵 (ℎ) = 2𝜋 𝑟 𝑝1 𝑟 𝑝2 (𝑟 𝑝1 +𝑟 𝑝2 ) 𝜆 𝐴𝐵 ∆𝐺 (ℎ=ℎ 0 ) 𝐴𝐵 𝑒 ( ℎ 0 -ℎ 𝜆 𝐴𝐵 ) ( 6
)
where λAB is the correlation length, chosen as 1.65 nm, according to Valsesia et al. (2018), and h0 is the minimum distance of separation between the particle and the surface, taken as 0.158 nm [START_REF] Valsesia | Direct Quantification of Nanoparticle Surface Hydrophobicity[END_REF] . The acidbase potential ΔG AB h=h0 is expressed as:

∆𝐺 (ℎ=ℎ 0 ) 𝐴𝐵 = -2 (𝛾 𝑝1 𝐴𝐵 + 𝛾 𝑝2 𝐴𝐵 -2√𝛾 𝑝1 𝐴𝐵 𝛾 𝑝2 𝐴𝐵 ) (7)
With 𝛾 𝑃1 𝐴𝐵 and 𝛾 𝑃2 𝐴𝐵 the polar component of the surface free energy for particles 1 and 2, respectively.

𝛾 𝑃 𝐴𝐵 was directly quantified using the method by Valsesia et al. (2018) and found to be equal to 33.91 and 31.82 mJ.m -2 for PSL COOH and NPT-P, respectively [START_REF] Valsesia | Direct Quantification of Nanoparticle Surface Hydrophobicity[END_REF] . Figure 1 shows that both particles are stable and low ionic strengths (5 mmol L -1 ). However, as ionic strength increases, the particles show differences in stability: PSL COOH is stable at high ionic strength (600 mmol L -1 ), while NPT-P aggregates, with dzH increasing from 359 to 623 nm in one hour. While the aggregation kinetics presented in Figure 1 took place at a pH of 6.5, the trends in stability were the same at pH 5 and 8, representing the pH range of natural waters (Figure S3). Based on these kinetics of aggregation, the NPT-P's critical coagulation concentration (CCC) in NaCl was determined to be 59 and 67 mmol L -1 NaCl at pH 6.5 and 8, respectively (Fig. S4). These values are lower than the CCC of 260 mmol L -1 (NaCl, unadjusted pH) previously determined by El Hadri et al. ( 2020) and show no significant increase in stability with pH [START_REF] El Hadri | Nanoplastic from Mechanically Degraded Primary and Secondary Microplastics for Environmental Assessments[END_REF] . This suggests that the NPT-P studied here has lower surface oxidation than those studied by El Hadri et al. (2020), as confirmed by a lower zeta (-33 vs. -44 mV). For NPT-P, the concentration of -COOH on the surface is lower, inhomogeneous, and uncontrolled. The mechanical degradation method used to produce NPT-P cannot control the -COOH functionalization of their surface, which induces possible variability on the CCC. The CCC value of PSL COOH was not assessed since these particles were stable up to 1 M NaCl, which is above environmentally relevant concentrations.

RESULTS AND DISCUSSION

Colloidal stability of nanoplastic models

The CCC of NPT-P was lower than that of PSL models, as illustrated in Table 2. The reasons behind differences in stability between our PSL and NPT-P models are discussed below. Differences in stability between PSL particles and environmentally relevant nanoplastic models are commonly observed. These recent observations confirm that particles' surface functionalization and morphology, as well as the presence of surfactants play key roles in the kinetics of aggregation. To characterize the effect of the particles' properties (size, surface potential, etc.) on their stability, the level of repulsion (energy barrier) between particles can be modeled by the extended Derjaguin Landau Verwey Overbeek (XDLVO) theory. According to the XDLVO theory, the interaction energies between NPT-P and PSL COOH are not significantly different (Table S1 and Figure S5). This demonstrates that size, surface charge, and hydrophobicity (Table 1), which are used to calculate interaction energy profiles, do not explain differences in stability. Instead, it suggests that NPT-P's morphologies and polydispersity (Table 1 and Figure S1), as well as the lack of surfactants in the dispersion, are responsible for the aggregation rates observed. Indeed, particle morphology will affect their attachment efficiency. NPT-P has an aspect ratio of 1.70 ± 0.57 and asperities on their surface, whereas PSL COOH has an aspect ratio of 1.02 ± 0.05 and a smooth surface. At close approach, particles with elongated shapes (high aspect ratios) have larger van der Waals attraction when their major axii face each other [START_REF] Vold | Van Der Waals' Attraction between Anisometric Particles[END_REF] . While this has been difficult to demonstrate experimentally [START_REF] Baalousha | Effect of Nanomaterial and Media Physicochemical Properties on Nanomaterial Aggregation Kinetics[END_REF] , a few aggregation experiments support this theory [START_REF] Dong | Aggregation Kinetics of Fragmental PET Nanoplastics in Aqueous Environment: Complex Roles of Electrolytes, PH and Humic Acid[END_REF][START_REF] Zhou | Role of Morphology in the Aggregation Kinetics of ZnO Nanoparticles[END_REF] . Also, the collision of irregular and rough particles is likely to occur between particle protrusions and edges [START_REF] Wu | Stability versus Flocculation of Particle Suspensions in Water-Correlation with the Extended DLVO Approach for Aqueous Systems, Compared with Classical DLVO Theory[END_REF] .

For this reason, it is more accurate to model the interaction energy between NPT-P and asperities, using the smallest radius of curvature as the asperity radius [START_REF] Lan | Unexpected Stability of Aqueous Dispersions of Raspberry-like Colloids[END_REF] . This significantly reduces the level of repulsion (Fig. S5) since the final volume of interaction is reduced and repulsive forces (electrostatic and acid-base) decay more quickly with distance than attractive forces (Lifshitz-van der Waals) [START_REF] Delrio | The Role of van Der Waals Forces in Adhesion of Micromachined Surfaces[END_REF][START_REF] Huang | Is Surface Roughness a "Scapegoat" or a Primary Factor When Defining Particle-Substrate Interactions?[END_REF] . Secondly, particle collision rate during perikinetic aggregation (i.e., induced by collisions driven by the Brownian motion) is always more significant for dispersions containing different particle sizes [START_REF] Petosa | Aggregation and Deposition of Engineered Nanomaterials in Aquatic Environments: Role of Physicochemical Interactions[END_REF] . Finally, NPT-P particles are free of surfactants, which have a stabilizing effect [START_REF] Petosa | Aggregation and Deposition of Engineered Nanomaterials in Aquatic Environments: Role of Physicochemical Interactions[END_REF][START_REF] Goodwin | Colloids and Interfaces with Surfactants and Polymers: An Introduction[END_REF] . Since the more environmentally relevant nanoplastic model, NPT-P, is not stable at high ionic strengths, the stabilizing effect of natural organic matters (NOMs) was studied.

Stabilization of NPT-P by natural organic matters

Two different NOMs were chosen to represent the wide variety of physico-chemical properties of naturally occurring organic matters. The interaction of the NPT-P with the natural organic matter was described by characterizing the size of NPT-P with NOM at high ionic strength. Asymmetrical flow field-flow fractionation coupled to static light scattering (A4F-SLS) was used to characterize the assembly of NPT-P with NOM as this technique can discriminate different size populations and changes in polydispersity. Figure 2 illustrates the fractograms obtained for NPT-P in the presence of HA (Fig 2a) and SA (Fig. 2b). NPT-P and HA alone have similar times of elution and variation of their radii of gyration (Rg) over time. For NPT-P with HA, the peak's elution time range is identical, suggesting that NPT-P and HA stay dispersed and retain their initial sizes. However, the maximum of the peak increases from 26 to 28 minutes, with the corresponding Rg increasing from 100 nm up to 270 nm. This shift suggests that a specific size fraction of HA is associated with NPT-P and formed larger heteroaggregates. Concerning the mixture of NPT-P with SA (Figure 3b), no fractograms were obtained for SA in these fractionation conditions due to its low scattering properties at this concentration (57 mg L -1 ). In the presence of SA, two peaks are observed: one eluted around 23 minutes and another around 26 minutes. The first peak corresponds to a Rg > 400 nm, while the second corresponds to a smaller Rg, around 250 nm. In A4F, the normal elution mode occurs when the relative diffusion between the different populations through the channel's height allows their separation according to the parabolic profile of the main velocity flow. However, an earlier peak with a high Rg is indicative of steric elution mode [START_REF] Messaud | An Overview on Field-Flow Fractionation Techniques and Their Applications in the Separation and Characterization of Polymers[END_REF] . In this mode, the particles become too large to be separated based on diffusion coefficient and are instead eluted by dragging forces. This first peak, in steric mode, corresponds to SA bridging separate NPT-P particles and sorbing onto small aggregates of NPT-P. The second peak overlaps with that of NPT-P alone and can be explained by the association of SA with single NPT-P or smaller aggregates. To validate the variations in size populations and distributions, Figure 3 shows the size distributions of NPT-P with HA and with SA at high ionic strength based on the Sparse Bayesian Learning (SBL) algorithm. This algorithm allows investigating differences in size population within the limits of the DLS resolution. With HA, the NPT-P size distribution is large but still covering the size range of the initial NPT-P. However, HA induces a shift towards a higher dH of 530 nm compared to the initial dH of 320 nm for NPT-P alone. This shift may be due to the non-covalent adsorption of larger HA molecules onto the NPT-P surface. In the presence of SA, the size distribution is less polydisperse but with a maximum dH around 820 nm. This larger size population can be explained by the physical association of SA with several (n> 2) NPT-P particles. The SBL algorithm reveals the most probable continuous size distribution among a set of 25 solutions.

The A4F-SLS fractograms and DLS size distributions (Figures 2 and3) suggest that HA and SA have different stabilization mechanisms. On the one hand, the HA molecules that are free in solution stabilize

NPT-P by providing electrostatic repulsion. Some low molecular weight HA molecules are also adsorbing onto the NPT-P surface and providing steric repulsion. The co-occurrence of these two mechanisms is supported by the fact that leonardite humic acid is one of the more large and polydisperse Hydrodynamic Diameter (nm)

NPT-P NPT-P + HA NPT-P + SA humic acids [START_REF] Beckett | Determination of Molecular Weight Distributions of Fulvic and Humic Acids Using Flow Field-Flow Fractionation[END_REF] . Indeed, electrostatic repulsion is attributed to the smaller size fraction of humic and fulvic acids [START_REF] Wu | Effect of Salinity and Humic Acid on the Aggregation and Toxicity of Polystyrene Nanoplastics with Different Functional Groups and Charges[END_REF][START_REF] Zhang | Aquatic Behavior and Toxicity of Polystyrene Nanoplastic Particles with Different Functional Groups: Complex Roles of PH, Dissolved Organic Carbon and Divalent Cations[END_REF] , while surface adsorption of the larger size fraction of HAs may occur via π-π interactions (with the aromatic structures of HA) and result in steric hindrance [START_REF] Li | Interactions between Nano/Micro Plastics and Suspended Sediment in Water: Implications on Aggregation and Settling[END_REF][START_REF] Singh | Understanding the Stability of Nanoplastics in Aqueous Environments: Effect of Ionic Strength, Temperature, Dissolved Organic Matter, Clay, and Heavy Metals[END_REF][START_REF] Yu | Aggregation Kinetics of Different Surface-Modified Polystyrene Nanoparticles in Monovalent and Divalent Electrolytes[END_REF][START_REF] Yu | Interactions between Engineered Nanoparticles and Dissolved Organic Matter: A Review on Mechanisms and Environmental Effects[END_REF] . Saavedra et al. ( 2019) noted that both HA and SA [START_REF] Saavedra | Influence of Nanoplastic Surface Charge on Eco-Corona Formation, Aggregation and Toxicity to Freshwater Zooplankton[END_REF] stabilized negatively charged particles. Due to HA's compact structure, adsorption of HA onto colloids did not increase their size. However, SA's high molecular weight (282 kDa, Fig. S6) and semi-rigid chains can lead to the formation of larger aggregates [START_REF] Buffle | A Generalized Description of Aquatic Colloidal Interactions: The Three-Colloidal Component Approach[END_REF] . Indeed, SA chains stabilize NPT-P particles by wrapping around single particles and small aggregates and bridging separate particles. These hetero-aggregates are then prevented from further aggregating by steric hindrance. Since SA is highly hydrophilic, its adsorption onto nanoplastics can be attributed to hydrogen bonds and van der Waals interactions [START_REF] Summers | Agglomeration of Nano-and Microplastic Particles in Seawater by Autochthonous and de Novo-Produced Sources of Exopolymeric Substances[END_REF][START_REF] Flemming | The Biofilm Matrix[END_REF][START_REF] Chen | The Impact of Nanoplastics on Marine Dissolved Organic Matter Assembly[END_REF][START_REF] He | Influence of Biofilm on the Transport and Deposition Behaviors of Nano-and Micro-Plastic Particles in Quartz Sand[END_REF] . Indeed, Bhattacharya et al. ( 2010) demonstrated a significant affinity between negatively charged carboxylated PSL and negatively charged algae [START_REF] Bhattacharya | Physical Adsorption of Charged Plastic Nanoparticles Affects Algal Photosynthesis[END_REF] . This affinity has been attributed to hydrogen bonds forming between the cellulosic component of algae and the PSL. Finally, using TEM, it appears that NPT-P is embedded in SA, while HA does not seem to have such a strong affinity with the NPT-P surface (Fig. S7).

Colloidal stability of NPT-P according to the nature and concentrations of NOM

These two mechanisms of stabilization will have distinct impacts on nanoplastics' colloidal stability in aqueous media. Figure 4 illustrates the size variation of NPT-P in 600 mmol L -1 NaCl with either HA or SA. At 600 mmol L -1 NaCl, the size of NPT-P increases in the absence of NOM (Fig. 1). Figure 4 shows that HA can stabilize NPT-P immediately, while SA stabilizes NPT-P within 10 minutes. The final sizes obtained (i.e., 300 nm for HA and 560 nm for SA) corroborate that different interactions occur between NPT-P and the two NOMs. In the presence of HA, the dzH of NPT-P remains constant around 300 nm. HA is highly polydisperse with a colloidal fraction centered around 230 nm. So, for kinetics of NPT-P with HA, the dzH presented in Figure 4 is a combination of both the dzH of NPT-P (339 nm) and the dzH of HA (230 nm). Despite this significant contribution of HA to the DLS signal, DLS will rapidly detect if aggregation occurs since scattering is highly sensitive to increases in size [START_REF] Xu | Particle Characterization: Light Scattering Methods[END_REF] .

In the presence of SA, the size of NPT-P increases within the first 10 minutes and stabilizes around 560 nm. Without NOM, such an increase in size takes more than 30 minutes (Fig. 1).

Contrary to HA, 57 mg L -1 SA does not contribute to the DLS signal. Therefore this increase in size followed by a stabilization is explained by a rapid hetero-association between NPT-P (particles and/or aggregates) and SA. Instead of keeping all particles separate as HA does, the SA biopolymer wraps separate or slightly aggregated plastic nanoparticles. Alginate molecules have been observed to form a layer with an approximate thickness of 20 nm around positively charged PSLs [START_REF] Oriekhova | Heteroaggregation of Nanoplastic Particles in the Presence of Inorganic Colloids and Natural Organic Matter[END_REF] . Also, when studying the aggregation of NPs with the dissolved (< 0.22 µm) fraction of organic matter that is naturally present in seawater, Chen et al. ( 2018) obtained similar kinetics of hetero-aggregation, with a rapid increase in size followed by a plateau around micrometric sizes [START_REF] Chen | The Impact of Nanoplastics on Marine Dissolved Organic Matter Assembly[END_REF] .

The relative quantity of particles and aggregates present in the dispersions over time was estimated based on the hydrodynamic diameter (dzH), the intensity of scattered light (I), and a spherical form factor (P), as described in Supplemental Information (S7). At high ionic strength, results show that the relative concentration of NPT-P plummets without NOM (Fig. S8). The relative NPT-P particle concentration does not decrease in the presence of HA, indicating colloidal stabilization induced by electrostatic repulsion. On the contrary, with SA, the relative NPT-P particle concentration decreases significantly and then stabilizes. Such behavior can be explained by the hetero-association of SA with NPT-P leading to a final state where all the NPT-P are associated with SA. Consequently, the rate of collision is reduced because of (i) the low rate of diffusion of large aggregates, (ii) the reduced number of separate particles and aggregates, and (iii) the effective repulsion between SA-coated surfaces. To validate this hypothesis, Figure 5 illustrates the different kinetics of association according to NaCl and NOM concentration. Due to the nature of the kinetics of aggregation of NPT-P/SA in the presence of SA, two different slopes were compared: k0-10, which represents the fast rate of aggregation from 0 to 10 minutes, and k10-60 representing the plateau, observed from 10 to 60 minutes. Figure 5a shows that NPT-P and SA's association is always faster than the homo-aggregation of NPT-P for the whole range of the ionic strength investigated (5 to 770 mmol L -1 , pH 8). So, the aggregation kinetics are initially accelerated by SA, which sorbs onto and bridges NPT-P particles. Figure 5b shows no significant increase in the rate of the initial, fast aggregation rate (k10-60) as a function of SA concentration. This suggests that small SA concentrations are sufficient to cover and stabilize NPT-P.

The SA that remains free in the solution (non-adsorbed) may have a stabilizing effect by increasing the solution's electrostatic repulsion. This agrees with Summers et al. (2018), who observed that a low concentration (≤ 1 mg L -1 ) of EPS in a nanoplastic dispersion could play a dispersant effect [START_REF] Summers | Agglomeration of Nano-and Microplastic Particles in Seawater by Autochthonous and de Novo-Produced Sources of Exopolymeric Substances[END_REF] .

Figure 5c presents the aggregation rate of NPT-P with NOM as a function of ionic strength during the stable section of aggregation's kinetics. In the presence of NOM, all aggregation rates were lower than without NOM, except at 5 mmol L -1 NaCl, where there was no significant difference (Fig. 5c andS3). In the presence of HA, NPT-P's aggregation rate is not significantly different from 0 nm min -1 except at 26 and 770 mmol L -1 (p < 0.05). At 770 mmol L -1 , the aggregation rate increases, suggesting that HA is losing its stabilizing effect due to a strong electrostatic screening by this high ionic strength. In the presence of SA, the aggregation rate hovers around 0.4 nm min -1 , especially at higher ionic strengths. This suggests that HA may have a stronger stabilizing effect than SA. The NOM concentration in Fig. 5c was the minimum concentration required best stabilize the nanoplastic models at 600 mmol L -1 NaCl (cf: Fig. 5d). Figure 5d shows that at high ionic strength HA rapidly reduces [START_REF] Saleh | Influence of Biomacromolecules and Humic Acid on the Aggregation Kinetics of Single-Walled Carbon Nanotubes[END_REF] . The slightly more effective stabilizing capacity of HA compared to SA can be attributed to the fact that SA sorbs onto particles while HA causes repulsion between them. Indeed, the first mechanism is more likely to form flocs that are large enough to be affected by gravity.

Environmental Implications of NOM-NP interactions

The aggregation rates presented in Figure 5 were determined at pH 6.5 for HA and pH 8 for SA, as these pHs are representative of terrestrial and marine aquatic systems, respectively. Kinetic studies performed at pH 5, 6.5, and 8 for both NOMs show that the same stabilizing mechanisms operate in the pH range of natural waters (Fig. S9). At a given ionic strength, aggregation rates did not significantly differ with pH (p < 0.05). This minimal pH-dependency is to be expected since (i) NPT-P's stability is not pH-dependent, (ii) HA stabilizes these particles by electrostatic and steric repulsion, which only requires the NOM to be significantly negatively charged and unfolded, (iii) SA stabilizes NPT-P by sorption via hydrogen bonds and van der Waals attraction, which are operational in this range of pH.

The different stabilizing mechanisms are summarized in Figure 6. These mechanisms have been shown

to stabilize natural colloids [START_REF] Buffle | A Generalized Description of Aquatic Colloidal Interactions: The Three-Colloidal Component Approach[END_REF] as well as engineered nanomaterials [START_REF] Saleh | Influence of Biomacromolecules and Humic Acid on the Aggregation Kinetics of Single-Walled Carbon Nanotubes[END_REF][START_REF] Espinasse | Transport and Retention of Colloidal Aggregates of C 60 in Porous Media: Effects of Organic Macromolecules, Ionic Composition, and Preparation Method[END_REF][START_REF] Hyung | Natural Organic Matter (NOM) Adsorption to Multi-Walled Carbon Nanotubes: Effect of NOM Characteristics and Water Quality Parameters[END_REF][START_REF] Loosli | Towards a Better Understanding on Agglomeration Mechanisms and Thermodynamic Properties of TiO2 Nanoparticles Interacting with Natural Organic Matter[END_REF][START_REF] Chen | Aggregation Kinetics of Alginate-Coated Hematite Nanoparticles in Monovalent and Divalent Electrolytes[END_REF] . When studying the stability of fullerene, Espinasse et al. (2007) also noted the different mechanisms of stabilization of HA and SA [START_REF] Espinasse | Transport and Retention of Colloidal Aggregates of C 60 in Porous Media: Effects of Organic Macromolecules, Ionic Composition, and Preparation Method[END_REF] .

Fullerene were more water-soluble in the presence of HA. HA's combination of hydrophobic regions and ionizable functional groups allows the former to sorb to hydrophobic particles and the latter to increase the particle's hydrophilicity. The amount of HA sorption onto carbon nanotubes was proportional to the HA aromaticity [START_REF] Hyung | Natural Organic Matter (NOM) Adsorption to Multi-Walled Carbon Nanotubes: Effect of NOM Characteristics and Water Quality Parameters[END_REF] . So, HA increases particles' stability by steric and charge stabilization. However, SA's large size promoted the aggregation of stable, polar fullerenes by bridging and encapsulating them [START_REF] Espinasse | Transport and Retention of Colloidal Aggregates of C 60 in Porous Media: Effects of Organic Macromolecules, Ionic Composition, and Preparation Method[END_REF] . Indeed, in NaCl, SA coats positively charged titanium dioxide and hematite nanoparticles which then confers electrostatic stability [START_REF] Loosli | Towards a Better Understanding on Agglomeration Mechanisms and Thermodynamic Properties of TiO2 Nanoparticles Interacting with Natural Organic Matter[END_REF][START_REF] Chen | Aggregation Kinetics of Alginate-Coated Hematite Nanoparticles in Monovalent and Divalent Electrolytes[END_REF] . SA also coats and stabilizes negatively charged, carbonaceous nanomaterials, such as single-walled carbon nanotubes [START_REF] Saleh | Influence of Biomacromolecules and Humic Acid on the Aggregation Kinetics of Single-Walled Carbon Nanotubes[END_REF] .

In aqueous media, we might expect that SA will stabilize NPs and increase their dispersal. At the same time, it will induce retention when it is attached to a solid interface (i.e., soils, sediments, etc.) [START_REF] He | Influence of Biofilm on the Transport and Deposition Behaviors of Nano-and Micro-Plastic Particles in Quartz Sand[END_REF][START_REF] Espinasse | Transport and Retention of Colloidal Aggregates of C 60 in Porous Media: Effects of Organic Macromolecules, Ionic Composition, and Preparation Method[END_REF] . Furthermore, Cunha et al. (2020) showed that nanoplastics and microplastics' presence enhanced the production of EPS carbohydrates by freshwater Cyanothece sp., suggesting a feedbackloop may occur [START_REF] Cunha | Microalgal-Based Biopolymer for Nano-and Microplastic Removal: A Possible Biosolution for Wastewater Treatment[END_REF] . The presence of NOM will impact nanoplastics environmental fate and affect the potential for co-transport of contaminants [START_REF] Liu | Polystyrene Nanoplastics-Enhanced Contaminant Transport: Role of Irreversible Adsorption in Glassy Polymeric Domain[END_REF][START_REF] Velzeboer | Strong Sorption of PCBs to Nanoplastics, Microplastics, Carbon Nanotubes, and Fullerenes[END_REF] . For example, compared to a matrix containing only a nanoplastic model and hydrophobic organic compounds (HOCs), the addition of HA to the matrix increased Daphnia magna's rate of uptake of the HOCs by ingestion [START_REF] Lin | Joint Effect of Nanoplastics and Humic Acid on the Uptake of PAHs for Daphnia Magna: A Model Study[END_REF] .

CONCLUSION

This study investigated the mechanisms of stabilization of nanoplastics by natural organic matter (NOM) according to the media's ionic strength. The interaction of the nanoplastics with NOMs was determined by characterizing the size distributions and shapes using asymmetrical flow field-flow fractionation coupled to static light scattering (A4F-SLS) and confirmed by dynamic light scattering (DLS).

According to their origin, the different NOM models, i.e., sodium alginate (SA) for marine environments and humic acid (HA) for terrestrial ones, present different stabilization mechanisms. Humic acid stabilizes the nanoplastic dispersion by electrostatic repulsion between particles, while larger molecules may sorb onto nanoplastics and provide a steric hindrance. However, sodium alginate adsorbs onto the nanoplastics' surface and bridges particles to form small aggregates that remain stable by steric hindrance against the increase in ionic strength. This study highlights the need to consider NOM's physico-chemical properties when assessing nanoplastics behavior in the aqueous environment.

SUPPORTING INFORMATION.

Images and size description of the nanoplastics models (Figure S1 Data was analyzed with ImageJ software and the NanoDefine plugin, using the watershed fitting mode (Verleysen et al., 2019). The length of the major axi and minor axi were the longest and shortest lengths of the minimum bounding rectangle. Based on these images and assuming the NPT-P particles is either a sphere or an ellipsoid, the specific surface area was determined to be 30.2 ± 16.4 and 34.2 ± 18.9 m 2 g -1 , respectively. The specific surface area was determined to be 29.6 ± 0.6 m 2 g -1 for PSL COOH.

The NPT-P minor and major axi followed a log-normal distribution, as determined with the orthogonal distance regression method. Equation (S1) was used to fit the data, with μ the average of distribution and σ the standard deviation of distribution.

𝑦 =

(

  Vasco-Flex, Cordouan Technologies, Pessac, France). The measured dH of an agglomerating suspension is the average of the dH of the individual particles and aggregates, weighted by their scattered light intensities[START_REF] Holthoff | Coagulation Rate Measurements of Colloidal Particles by Simultaneous Static and Dynamic Light Scattering[END_REF] . The backscattered light is collected at a geometric angle of 170° with respect to the incident beam direction. For time-resolved DLS, each correlation function was accumulated for 60 seconds and were spaced 30 seconds apart. DLS measurements of stock solutions are composed of an average of six measurements of 60 seconds. The z-average hydrodynamic diameter (dzH) was determined by fitting a normal distribution to the raw data using the cumulant algorithm. To analyze the different populations in size present in a dispersion, the Sparse Bayesian learning (SBL) algorithm was used. The distribution of the NPs' gyration radii was measured by static light scattering (DAWN HELEOS 18 Angles, Wyatt Technology) with prior size fractionation using an asymmetrical flow field flow fractionation (A4F, Eclipse 3+, Wyatt Technology, Dernbach, Germany) and a UV-vis absorbance detector (1200 series, Agilent Technologies, France) as a concentration detector at 254 nm. The global method of A4F separation was used. It was previously optimized and described byGigault et al. (2017) [START_REF] Gigault | Asymmetrical Flow Field Flow Fractionation Methods to Characterize Submicron Particles: Application to Carbon-Based Aggregates and Nanoplastics[END_REF]

Figure 1 : 5 (

 15 Figure 1: Aggregation kinetics of 4 mg L -1 a) PSL COOH and b) NPT-P in either 5 mmol L -1 or 600 mmol L -1 NaCl at pH 6.5 (Error bar = standard deviation)

  Indeed,Yu et al. (2019) determined that non-functionalized and carboxylated PSL spheres have a CCC of 310 and 308 mmol L -1 NaCl, respectively[START_REF] Yu | Aggregation Kinetics of Different Surface-Modified Polystyrene Nanoparticles in Monovalent and Divalent Electrolytes[END_REF] .Mao et al. (2020) found an even greater CCC of 591 mmol L -1 NaCl for non-functionalized PSL spheres[START_REF] Mao | Nanoplastics Display Strong Stability in Aqueous Environments: Insights from Aggregation Behaviour and Theoretical Calculations[END_REF] . The aging of these particles by UV-irradiation strongly oxidized their surface. This caused stronger electrostatic repulsion, and consequently, the CCC increased up to 1108 mmol L -1 . Singh et al. (2019) found a lower CCC of 140 mmol L -1 NaCl for nonfunctionalized PSL and attributed this difference to the removal of surfactants[START_REF] Singh | Understanding the Stability of Nanoplastics in Aqueous Environments: Effect of Ionic Strength, Temperature, Dissolved Organic Matter, Clay, and Heavy Metals[END_REF] . The CCC calculated for NPT-P was coherent with observations made on other non-spherical, non-emulsified and surfactantfree, nanoplastic models. For example, NPs produced by laser ablation of PS show strong aggregation in 300 mmol L -1 NaCl 39 . NPs produced from mechanical fragmentation polyethylene glycol terephthalate (PET-G) had a CCC of 54 mmol L -1 NaCl at pH 6, and 110 mmol L -1 NaCl at pH 10 43 .

Figure 2 :

 2 Figure 2: Fractograms showing absorbance (line) and radius of gyration (Rg) (points) of a) NPT-P in 5 mmol L -1 NaCl, NPT-P with 30 mg L -1 HA in 6oo mmol L -1 NaCl, and 30 mg L -1 HA in 600 mmol L -1 NaCl at pH 6.5 and b) NPT-P in 5 mmol L -1 NaCl and NPT-P with 57 mg L -1 SA in 600 mmol L -1 NaCl at pH 8, as a function of retention time

Figure 3 :

 3 Figure 3: Average of intensity-based size distributions according to SBL algorithm of NPT-P with 30 mg L -1 humic acid (HA) at pH 6.5 or 57 mg L -1 sodium alginate (SA) at pH 8, in 6oo mmol L -1 NaCl measured between 45 and 60 minutes (n ≥ 18).

Figure 4 :

 4 Figure 4: Aggregation kinetics of 4.0 mg L -1 NPT-P, in 600 mmol L-1 NaCl, with 57 mg L -1 sodium alginate (SA) at pH 8 and 30 mg L -1 humic acid (HA) at pH 6.5 (Error bar = standard deviation)

Figure 5 :

 5 Figure 5: a) Aggregation rate of NPT-P and fast aggregation rate (k0-10) of NPT-P with 57 mg L -1 SA, at pH 8 as a function of NaCl concentration b) fast aggregation rate (k0-10) of NPT-P with varying concentrations of SA at 600 mmol L -1 NaCl and pH 8. Aggregation rates of NPT-P with c) 57 mg L -1 SA (k10-60) at pH 8 and with 30 mg L -1 HA (k0-60) at pH 6.5 as a function of NaCl and d) in 600 mmol L -1 NaCl, with varying concentrations of HA at pH 6.5 and different concentrations of SA at pH 8.

  and S2, pages S2-S3); Description of the asymmetrical field flow fractionation method (Section S1, page S4); Aggregation rates and Critical Coagulation Concentrations of the nanoplastics in NaCl (Figure S3 and S4); Results of the Extended Derjaguin Landau Verwey Overbeek (XDLVO) model (TableS1and FigureS5, page S7,); Characterization of the sodium alginate (FigureS6, text page S8); Images of nanoplastics in the presence of natural organic matter (FigureS7and text page S9); Evolution of nanoplastics concentration according to time calculated from static light scattering (FigureS8, text page S10 and S11); Aggregation kinetics of nanoplastics models in the presence of natural organic matter (FigureS9, page S12). This material is available free of charge via the internet at http://pubs.acs.org.

Figure S2 :

 S2 Figure S2: Histogram of short axis (green) and long axis (purple), as determined by TEM images of a) PSL COOH and b) NPT-P nanoplastic models in linear distribution (PSL COOH: n = 212 and NPT-P: n = 283) TEM images were obtained by a Jeol JEM 2100 HR (200kV) with an LaB6 filament. The camera was a Gatan Orius SC 200 D. A 4 μL drop was deposited on a full carbon grid and allowed to air dry.

••

  For the NPT-P major axis: o μ = 395.70 ± 30.65 o σ = 380.52 ± 48.34 o A = 20734.02 ± 6774.76 o R 2 (COD) = 0.99994 For the NPT-P minor axis : o μ = 213.54 ± 3.93 o σ = 461.88 ± 13.53 o A = 9986.26 ± 260.98 o R 2 (COD) = 0.98101

Figure S3 :S7*

 S3 Figure S3: Aggregation rate of a) PSL COOH and b) NPT-P models, as a function of NaCl concentration and pH (Error bars = standard deviation)

Figure S5 :Figure S6 :

 S5S6 Figure S5: Interaction energy, scaled to kBT, between different nanoplastic models, according to XDLVO theory

Table 2 :

 2 Summary of different critical coagulation concentrations (CCC) of NaCl for various NP models

	NPs models studied
	Nomenclature : Composition Type of particle Surface	CCC (mmol L -1 )	Reference
	functionalization* Nominal size (Charge)
	PS Latex sphere NF 100nm (-)	310 in NaCl at pH 7.4
	PS Latex sphere COOH 100nm (-)	308 in NaCl at pH 7.4	Yu et al., 2019 ( 39 )
	PS Laser ablation 60 nm (-)	Not determined. Strong aggregation
		in 300 mmol L -1 NaCl pH 7.4
	PS Latex sphere NF 100 nm (-)	591 in NaCl pH 7.5
	PS Latex sphere aged by UV-irradiation during 60 hours NF 100 nm (-) **	957 in NaCl pH 7.5	Mao et al., 2020 ( 37 )
	PS Latex sphere aged by UV-irradiation during 120 hours NF 100 nm (-) **	1108 in NaCl pH 7.5
	PS Latex sphere NF 240(-)	140 in NaCl pH 6	Singh et al., 2019 ( 31 )
	PET-G Mechanical degradation 500 nm (-) 54 in NaCl pH 6 110 in NaCl pH 10	Dong et al., 2020 ( 43 )
	PS Mechanical degradation 350 nm (-)	260 in NaCl pH unadjusted	El Hadri et al., 2020 ( 48 )
	PS Mechanical degradation 350 nm (-)	59 in NaCl pH 6.5	NPT-P particles studied
	PS Mechanical degradation 350 nm (-)	67 in NaCl pH 8	here

*NF = Non-functionalized, COOH = carboxylated ** Initial particles were non-functionalized. Aging produced an increasing amount of carbonyl functional groups on the surface.

  NPT-P's attachment efficiency. This is supported by the observations made by Singh et al. (2019), indicating that low concentrations of HA increased the CCC of negatively charged PSL spheres almost 4-fold 31 . However, even high SA concentrations do not reduce k10-60 under 0.6 nm min -1 . This agrees with Summers et al. (2018), who show that high alginate concentrations can have a flocculant effect 42 . Furthermore, Lodeiro et al. (2016) noted that SA only slightly increased the stability of silver nanoparticles 72 . Saleh et al. (2010) also noted that HA was more effective than SA at stabilizing carbon nanotubes in NaCl

ACKNOWLEDGEMENTS

We acknowledge Julie Borgese for the illustrations and Ludivine Rault for the TEM images.

Insight into mechanisms

Alice Pradel a* , Séléna Ferreres a , Cloé Veclin b , Hind El Hadri b , Maud Gautier a , Bruno Grassl b , Julien Gigault a,c* a Univ Rennes, CNRS, Géosciences Rennes -UMR 6118, 35000 Rennes, France b CNRS/ Univ Pau & Pays Adour/ E2S UPPA, Institut des sciences analytiques et de physicochimie pour l'environnement et les matériaux, UMR 5254, 64000, Pau, France c TAKUVIK laboratory, CNRS/Université Laval, 1045, av. de la Médecine, Québec (Québec) G1V 0A6 Canada *alice.pradel@univ-rennes1.fr; * julien.gigault@takuvik.ulaval.ca

The Supporting Information contains 9 figures, 1 table, 2 sections of text (SI1 and SI2), 9 references, 13 pages. The mobile phase was composed of 0.5 mmol L -1 NaNO3 (>99% purity Reagent Plus, Sigma Aldrich), which was filtered on polyethersulfone (PES) filters (0.1 μm, Pall®), purchased from VWR (Fontenay-sous-Bois, France). The injection volume was 100 µL. First, the effects of the duration of the focus period and the ionic strength of the injected dispersion, on the quality of detection were studied.

Table of contents:

Subsequently, a focus time of 5 minutes and an ionic strength of 600 mmol L -1 was selected. The injected dispersion was prepared in the same way as for the aggregation kinetic study, described below.

Data from a minimum of 14 out of 18 SLS detectors were collected and processed using Astra software, version 6 (Wyatt Technology). The radius of gyration (Rg) was determined using the Berry formalism using SLS signal at different angles. The relative particle concentration (n/n0) was determined according to the relation that links the intensity of scattered light and the particle size. This relationship is only valid assuming that all particles are spherical (Wyatt, 1993). The intensity of scattered light, at a give scattering angle and particle concentration I(ϴ,c), is given by the following equation:

with ϴ, the angle at which light is scattered, which is equal to 170 degrees and c the concentration in g L -1 , K the instrument constant, MW the molar mass in g mol -1 and Pϴ the form factor. For a sphere, the form factor Pϴ is given by:

For a sphere, u is defined as:

with rp the sphere's radius and q the wave vector, defined as: