Nurul I Supiandi

G Charron

M Tharaud

L Cordier

J.-M Guigner

M F Benedetti

Y Sivry

Isotopically labelled nanoparticles at relevant concentrations: how low can we go? -The case of CdSe/ZnS QDs in surface waters

Keywords: detection limits, nanoparticles, isotopic labelling, quantum dots, aquatic matrices, HR-ICP-MS

Analytical barriers impose to work at nanoparticles (NPs) concentration orders of magnitude higher than the expected NPs concentrations in the environment. To overcome these limitations, the use of non-traditional stable isotope tracers incorporated in NPs (spiked-NPs) coupled with HR-ICP-MS has been proposed. The performance and efficiency of this analytical method was assessed in the case of quantum dots (QDs). Multi-isotopically labelled 111 Cd 77 Se/ 68 ZnS QDs were synthesized and their dissemination in natural aquatic matrices (river, estuarine and sea waters) was modelled at very low concentrations (from 0.1 to 5000 ppt). The QD limits of quantification (QD-LOQ) in each matrix were calculated according to the isotopic tracer.

In ultrapure and simple medium (HNO3 2%), Zn, Cd and Se originated from the QDs were quantifiable at concentrations of 10, 0.3 and 6 ppt, respectively, which is lower than the conventional HR-ICP-MS LOQs. In aquatic matrices, the QD-LOQs increase 10-, 130-, and 250fold for Zn, Cd, and Se, respectively, but remain relevant of environmental concentrations (3.4 ppt ≤ QD-LOQs ≤ 2.5 ppb). These results validate the use of isotopically-labelled ENPs at relevant concentrations in experimental studies related to either their fate, behavior or toxicity in most aquatic matrices.

INTRODUCTION

Engineered nanoparticles (ENPs) are widely used in various consumer products due to their exceptional chemical, optical, magnetic or mechanical properties. [START_REF] Mitrano | Review of Nanomaterial Aging and Transformations through the Life Cycle of Nano-Enhanced Products[END_REF] To name a few, TiO2 NPs are used as photocatalyst in water and air treatment, [START_REF] Lan | Mini Review on Photocatalysis of Titanium Dioxide Nanoparticles and Their Solar Applications[END_REF] or as whitening agent in paints and in food and food packaging products, [START_REF] Contado | Nanomaterials in Consumer Products: A Challenging Analytical Problem[END_REF][START_REF] Calzolai | Measuring Nanoparticles Size Distribution in Food and Consumer Products: A Review[END_REF][START_REF] Mattarozzi | Analytical Approaches for the Characterization and Quantification of Nanoparticles in Food and Beverages[END_REF] Ag NPs are added to fabrics for anti-bacterial purpose, [START_REF] Yetisen | Nanotechnology in Textiles[END_REF][START_REF] Chernousova | Silver as Antibacterial Agent: Ion, Nanoparticle, and Metal[END_REF][START_REF]Samsung Silver Nano Technology, Refigerator Silver Nano, Washing Machine Silver Nano[END_REF][START_REF] Mitrano | Tracking Dissolution of Silver Nanoparticles at Environmentally Relevant Concentrations in Laboratory, Natural, and Processed Waters Using Single Particle ICP-MS (SpICP-MS)[END_REF][START_REF] Kühn | Investigation of Coatings of Natural Organic Matter on Silver Nanoparticles under Environmentally Relevant Conditions by Surface-Enhanced Raman Scattering[END_REF][START_REF] Ibrahim | Characterization and Antimicrobial Properties of Cotton Fabric Loaded with Green Synthesized Silver Nanoparticles[END_REF][START_REF] Mackevica | The Release of Silver Nanoparticles from Commercial Toothbrushes[END_REF] ZnO NPs act as UV absorbers in sunscreens, [START_REF] Tyagi | Comparative Analysis of the Relative Potential of Silver, Zinc-Oxide and Titanium-Dioxide Nanoparticles against UVB-Induced DNA Damage for the Prevention of Skin Carcinogenesis[END_REF][START_REF] Wiechers | Engineered Inorganic Nanoparticles and Cosmetics: Facts, Issues, Knowledge Gaps and Challenges[END_REF][START_REF] Newman | The Safety of Nanosized Particles in Titanium Dioxide-and Zinc Oxide-Based Sunscreens[END_REF] and CeO2 NPs are incorporated in catalytic converters in the automotive industry. [START_REF] Senanayake | Unique Properties of Ceria Nanoparticles Supported on Metals: Novel Inverse Ceria/Copper Catalysts for CO Oxidation and the Water-Gas Shift Reaction[END_REF] Quantum dots (QDs), namely fluorescent nanocrystals made of semiconducting materials, are used in lightning and light display devices for color and brightness enhancement, in solar panels and bio-sensors. [START_REF] Dai | Quantum-Dot Light-Emitting Diodes for Large-Area Displays: Towards the Dawn of Commercialization[END_REF][START_REF] Nann | Quantum Dots for Electro-Optic Devices[END_REF][START_REF] Pickering | Patterned Mist Deposition of Tri-Colour CdSe/ZnS Quantum Dot Films toward RGB LED Devices[END_REF][START_REF] Shirasaki | Emergence of Colloidal Quantum-Dot Light-Emitting Technologies[END_REF][START_REF] Kamat | Quantum Dot Solar Cells. The Next Big Thing in Photovoltaics[END_REF][START_REF] Chuang | Improved Performance and Stability in Quantum Dot Solar Cells through Band Alignment Engineering[END_REF] The use and disposal of these products leads to the release of nanomaterials in wastewater or in the environment (e.g. surface waters, soils).

Surface waters which are known as one of the major vectors of ENPs dispersion in environment, [START_REF] Gottschalk | Modeled Environmental Concentrations of Engineered Nanomaterials (TiO2, ZnO, Ag, CNT, Fullerenes) for Different Regions[END_REF] have inspired numerous publications aiming at determining ENPs fate and behavior in surface waters, in particular through assessing their colloidal and chemical stabilities. [START_REF] Domingos | Aggregation of Titanium Dioxide Nanoparticles: Role of a Fulvic Acid[END_REF][START_REF] Collin | Environmental Release, Fate and Ecotoxicological Effects of Manufactured Ceria Nanomaterials[END_REF][START_REF] Sivry | Behavior and Fate of Industrial Zinc Oxide Nanoparticles in a Carbonate-Rich River Water[END_REF][START_REF] Gelabert | Uncoated and Coated ZnO Nanoparticle Life Cycle in Synthetic Seawater[END_REF][START_REF] Conway | Dissolution, and Transformation of Copper Nanoparticles in Natural Waters[END_REF][START_REF] Rocha | The Fate of Polyol-Made ZnO and CdS Nanoparticles in Seine River Water (Paris, France)[END_REF][START_REF] Furtado | Monitoring the Fate and Transformation of Silver Nanoparticles in Natural Waters[END_REF] However, these physico-chemical studies were often conducted at ENPs concentrations that are orders of magnitude higher than the levels expected from their dissemination in aquatic environments. The concentration of ENPs has been shown to affect their dissolution and their aggregation state, thereby potentially impacting their behavior. For instance, the increase in ZnO NPs concentrations decreased their dissolution, [START_REF] Yung | Salinity-Dependent Toxicities of Zinc Oxide Nanoparticles to the Marine Diatom Thalassiosira Pseudonana[END_REF] whereas their homoaggregation increased with increasing concentrations. [START_REF] Keller | Stability and Aggregation of Metal Oxide Nanoparticles in Natural Aqueous Matrices[END_REF] Therefore, working at environmentally relevant concentrations is critical when studying the fate and behavior of ENPs in natural systems.

Nevertheless, working at realistic concentrations also means overcoming two common analytical barriers: i) unsuitable instrumental detection limits and ii) the geochemical environmental background, i.e. the natural occurrence of some constitutive elements of the ENPs in the environment. Recent development of methods such as single counting ICP-MS (sp-ICP-MS) allow the detection of NPs with limit of detections achieving ppt levels. However, when studying the behavior (e.g. dissolution) of a type of NPs in an environmental medium e.g. river/seawater containing already the same constitutive elements as the NPs, sp-ICP-MS will not be able distinguishing between natural and anthropogenic nanomaterial. Hence, recent studies have proposed the use of non-traditional stable isotopes to overcome these analytical barriers, i.e. the use of isotopically modified or "spiked" ENPs which can be quantified by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). [START_REF] Dybowska | Synthesis of Isotopically Modified ZnO Nanoparticles and Their Potential as Nanotoxicity Tracers[END_REF][START_REF] Buffet | Fate of Isotopically Labeled Zinc Oxide Nanoparticles in Sediment and Effects on Two Endobenthic Species, the Clam Scrobicularia Plana and the Ragworm Hediste Diversicolor[END_REF][START_REF] Larner | Tracing Bioavailability of ZnO Nanoparticles Using Stable Isotope Labeling[END_REF][START_REF] Khan | Stable Isotope Tracer To Determine Uptake and Efflux Dynamics of ZnO Nano-and Bulk Particles and Dissolved Zn to an Estuarine Snail[END_REF][START_REF] Laycock | Earthworm Uptake Routes and Rates of Ionic Zn and ZnO Nanoparticles at Realistic Concentrations, Traced Using Stable Isotope Labeling[END_REF] For instance, this strategy was employed to determine the bio-uptake of TiO2, Ag, and ZnO NPs in aquatic organisms, using [START_REF] Mccurdy | Improving ICP-MS Analysis of Samples Containing High Levels of Total Dissolved Solids[END_REF] Ti, 109 Ag, 67 Zn tracers, respectively. [START_REF] Dybowska | Synthesis of Isotopically Modified ZnO Nanoparticles and Their Potential as Nanotoxicity Tracers[END_REF][START_REF] Bourgeault | The Challenge of Studying TiO2 Nanoparticle Bioaccumulation at Environmental Concentrations: Crucial Use of a Stable Isotope Tracer[END_REF][START_REF] Croteau | Isotopically Modified Silver Nanoparticles to Assess Nanosilver Bioavailability and Toxicity at Environmentally Relevant Exposures[END_REF] Quantitative detection of the ENPs was achieved at concentrations as low as 5 ppb, 6 ppt, and 1 ppm for 47 TiO2, 109 Ag, and 67 ZnO NPs, respectively, showing the advantages of ENPs isotopic labelling. Although the necessity to work at realistic concentrations has been recently discussed, [START_REF] Laycock | Earthworm Uptake Routes and Rates of Ionic Zn and ZnO Nanoparticles at Realistic Concentrations, Traced Using Stable Isotope Labeling[END_REF][START_REF] Bourgeault | The Challenge of Studying TiO2 Nanoparticle Bioaccumulation at Environmental Concentrations: Crucial Use of a Stable Isotope Tracer[END_REF][START_REF] Croteau | Isotopically Modified Silver Nanoparticles to Assess Nanosilver Bioavailability and Toxicity at Environmentally Relevant Exposures[END_REF] the exposure concentrations generally used in many ENPs fate and behavior studies remain higher than the estimated ones in natural media and, so far, none has tried to accurately determine to which extent the labelling of ENPs with stable isotopes would allow to work at the cutting edge of analytical barriers in ENPs fate and behavior studies.

In the present study, we aimed to determine for the first time the analytical limits of the ENP isotopic labelling technique in aquatic matrices (river, estuarine, and seawaters). Quantum dots with a CdSe/ZnS core/shell structure enriched in 111 Cd, 77 Se, and 68 Zn (hereafter called multispiked QDs) were synthesized for the first time in this study. Even though the estimated QDs concentration in aquatic environment is less than 1 ppt, [START_REF] Gottschalk | Modeling Flows and Concentrations of Nine Engineered Nanomaterials in the Danish Environment[END_REF][START_REF] Piccinno | Industrial Production Quantities and Uses of Ten Engineered Nanomaterials in Europe and the World[END_REF] their increasing environmental release will unavoidably follow due to the increasing presence of QDs in consumer products, especially electronic devices (e.g. TV set or light bulbs) -therefore, CdSe/ZnS QDs make for a worthy analytical case. As a representative example of core/shell-structured nanomaterials, their reactivity is expected to be more complex than that of homogeneous NPs. These ENPs make for a challenging system on which to gauge the analytical limits of the isotopic labelling strategy. These multispiked QDs were then dispersed at very low and environmentally relevant concentrations (ppt-ppb) into the chosen aquatic matrices. Measurements by HR-ICP-MS and subsequent chemometric processing allowed to accurately determine the limits of quantifications according to the element labelled and to the matrix composition (QD-LOQ). Chemometrics was proven to be an efficient analytical approach e.g. during method optimization. [START_REF] Majedi | Chemometric Analytical Approach for the Cloud Point Extraction and Inductively Coupled Plasma Mass Spectrometric Determination of Zinc Oxide Nanoparticles in Water Samples[END_REF] This work is expected to provide valuable basis to any future study aiming at using isotopically labelled ENPs to determine their fate, behavior, or toxicity in natural media while working at relevant concentrations.

MATERIALS AND METHODS

Composition of aquatic matrices

The Seine river water was chosen first, as it is highly representative of both a natural carbonaterich system and a highly anthropized watershed, which is relevant next to the possibility of an accidental release of ENPs. The Seine river water sample was collected behind the University Pierre et Marie Curie (Quai Saint-Bernard, Jardin Tino Rossi, Paris, France, 48.8475° N, 2.3614° E), and filtered through a 0.2 µm cellulose acetate membrane, stored in pre-cleaned, acid-washed polyethylene bottles and divided in three subsamples for: i) major and trace elemental analyses, where the aliquot was acidified with 15 N distilled nitric acid to obtain 2% of nitric acid in the samples, ii) analysis of anions (Cl -, SO4 2-, NO3 -), and iii) the determination of isotopic labelling limits experiments. Sampling and analytical procedures were described by Benedetti et al. [START_REF] Benedetti | Chemical Weathering of Basaltic Lava Flows Undergoing Extreme Climatic Conditions: The Water Geochemistry Record[END_REF] The chemical composition of the Seine water sample is displayed in Table S1.

The second surface water chosen was a synthetic seawater (SW), prepared following the American Society for Testing and Materials (ASTM) International guidelines (Table S2).

Dissolved Zn, Cd and Se with natural isotopic abundances were also added to this SW at concentrations provided by Seine Normandie Water Agency. [START_REF]Etude 2008 -Guide toxique[END_REF] Afterwards, from the precedent Seine river water and SW, three different solutions of estuarine waters were prepared by mixing the Seine river water sample and the SW solution (V/V) by the ratios 75:25 (estuarine water 1), 50:50 (estuarine water 2), and 25:75 (estuarine water 3). All waters were stored at 4°C in pre-cleaned, acid-washed polyethylene bottles.

Synthesis of multi-isotopically labelled CdSe/ZnS quantum dots

All chemical products with natural isotopic composition were purchased from Sigma Aldrich: sulfur (S, 99.9%, powder), oleic acid (OA, technical, 90%), trioctylphosphine (TOP, 90%), 1octadecene (ODE, 90%), chloroform (CHCl3, 99%), and thioglycolic acid (TGA, 99%). Chemicals with modified isotopic composition were purchased from ISOFLEX USA: cadmium oxide (CdO powder) enriched at 96.00% in 111 Cd, zinc oxide (ZnO powder) enriched at 99.16% in 68 Zn, and selenium (Se powder) enriched at 99.20% in 77 Se.

A common route towards CdSe/ZnS nanocrystals consists in nucleating CdSe cores by quickly injecting a chalcogenide mixture into a hot organic solution of the metal salts in a process known as the hot injection method. The growth of the ZnS shell is either achieved in a subsequent step or one-pot in the same step as nucleation, as described by Bae et al. [START_REF] Bae | Single-Step Synthesis of Quantum Dots with Chemical Composition Gradients[END_REF] In this protocol, cadmium oxide, zinc acetate, elemental selenium and elemental sulfur were used as the metal and chalcogen precursors, respectively. Transposing this route to multi-spiked CdSe/ZnS QDs raised difficulties regarding the availability of isotopically-modified precursors of suitable isotopic enrichments. 111 CdO and 77 Se powders were commercially available with 111 Cd and 77 Se abundances markedly different from the natural ones (96.00% and 99.20%, respectively) that could impart a high isotopic contrast to the QDs compared to the natural background. However, zinc acetate was commercially available with a 48% 68 Zn abundancy only, which did not contrast enough with the 18% natural abundancy to support detection of the spiked QDs in the ppt range in natural matrices. Therefore, Bae's protocol was adapted to start from ZnO as the zinc precursor, as it was commercially available with 99.16% enrichment in 68 Zn.

Multi-spiked CdSe/ZnS QDs were synthesized by the hot injection method in octadecene following an adaptation of the protocol from Bae et al. [START_REF] Bae | Single-Step Synthesis of Quantum Dots with Chemical Composition Gradients[END_REF] The modification consisted in substituting 68 Zn-enriched ZnO for the initial zinc acetate precursor which was not available with a suitable modified isotopic composition. [START_REF] Bae | Single-Step Synthesis of Quantum Dots with Chemical Composition Gradients[END_REF] Briefly, 51.364 g (0.4 mmol) of 111 Cd-enriched CdO, 325.56 g (4 mmol) of 68 Zn-enriched ZnO, 6.746 g (23.88 mmol) of oleic acid, 20 mg (0.2 mmol) of succinic anhydride and 23 mL octadecene were placed in a 100 mL three-necked round bottom flask equipped with a temperature probe, a coil condenser and connected to an inert lane. The flask was nested into a heating mantel whose power was controlled by a temperature controller connected to the temperature probe. The mixture was degassed under 10 mbar, heated to 150°C and maintained under these conditions for 20 min. Next, the montage was filled with N2 and further heated to 180°C for 10 min and then 250°C for 10 more minutes to fully dissolve the ZnO powder. The temperature was then increased to 310°C. At this temperature, a mixture of 31.584 mg (0.4 mmol) of 77 Se-enriched selenium and 128.28 mg (4 mmol) sulfur dissolved in 3 mL of trioctylphosphine under N2 atmosphere was quickly injected into the reaction flask under vigorous stirring.

Immediately after injection, the temperature was lowered to 300°C and the flask was kept at this temperature for 15 min to promote the growth of the nanocrystals. The flask was then cooled down to room temperature. The QDs were precipitated with 100 mL of acetone by centrifugation at 6000 rpm and then purified to remove excess reactants by redispersion-precipitation cycles twice with 90 mL acetone and once with 40 mL methanol.

Then, water-soluble QDs were prepared by replacing the oleic acid ligands attached to the surface of the QDs with thioglycolic acid (TGA). In a 100-mL round bottom flask, a solution of 10 mL of chloroform and 1.6 mL of thioglycolic acid (TGA) was prepared. Next, the flask was nested into an ultrasonic bath and 460 µL of the multi-spiked QDs stock solution were added under sonication and maintained under sonication for 1 min. The flask was then equipped with a condenser and transferred into a water bath. The reaction mixture was stirred using a stir bar and heated under reflux for 2 hours. After cooling to room temperature, the TGA-coated QDs were precipitated by centrifugation at 6000 rpm and then purified by redispersion-precipitation cycles twice with 10 mL chloroform and once with 10 mL acetone. Finally, the TGA-coated QDs were dispersed in pH 8 borate buffer and purified using Centricon® Centrifugal Filter Units (30kD MWCO) by exchanging the solvent once with a fresh portion of pH 8 borate buffer and 4 times with ultrapure water adjusted at pH 10. The final solution had a volume of 10 mL.

Quantum dots characterization

Optical characterization of QDs was carried out by measuring the UV-vis absorption spectra of the TGA-coated QDs stock solution with Thermo Scientific Evolution™ 600 UV-vis spectrometer.

The fluorescence signal was collected using the Horiba Scientific FluoroMax®-4 spectrofluorometer.

Diluted suspensions QDs were deposited on copper grid to observe their sizes and shapes by Transmission Electron Microscopy (TEM), using a JEOL 2100F electron microscope operating at 200 kV and equipped with a field emission gun, a high-resolution UHR pole piece and a Gatan GIF 200l imaging filter. To perform chemical analysis, this microscope was coupled with electrondispersive X-ray spectroscopy (EDXS) using a JEOL detector with an ultrathin window allowing detection of low atomic mass elements. TEM pictures were analysed with the software ImageJ 1.51n.

The total concentrations of Cd, Se, and Zn in the TGA-coated QDs stock solution were measured by ICP-OES (Thermo Scientific iCAP 6000 Series) after a complete acid digestion with HF/HNO3 (see protocol in SI) and then after simple acidification (2% of nitric acid) to evaluate the necessity of using complete acid digestion for all experimental samples.

Model dissemination of QDs in aquatic matrices

All materials used in these experiments (PP tubes, bottles and pipette tips) were washed with HCl 1 N to eliminate possible contaminations in trace metals, especially Zn, Cd and Se.

TGA-coated CdSe/ZnS quantum dots were added separately in all 5 selected natural surface waters, at target concentrations varying from 0.1 to 5000 ppt of Zn issued from the QDs, resulting in 0.03 to 1500 ppt in Cd and 0.02 to 1000 ppt in Se. These choices are in the lowest relevant range of the ENPs estimated concentrations in surface waters (ppt to ppb levels). [START_REF] Gottschalk | Modeling Flows and Concentrations of Nine Engineered Nanomaterials in the Danish Environment[END_REF] The QDs were also added, with the same target concentrations, in both HNO3 2% and NaNO3 0.01M, used as control media, since there is no occurrence of natural Zn, Cd and Se in both solutions, and the NaNO3 0.01 M solution is representative of the highest typical ionic strength found in river and estuarine waters. [START_REF] Garrels | Minerals, and Equilibria[END_REF] All samples were triplicated to assess the experimental reproducibility.

After QDs addition to these media, all samples were agitated then acidified to obtain 2% of nitric acid in the samples, then left overnight prior to dilution and analysis. Simple acidification was sufficient to completely dissolve the QDs, thus the complete acid digestion with HF/HNO3 was not further performed. The acidification step is representative of the final step used in many studies prior to ENPs analysis. [START_REF] Sivry | Behavior and Fate of Industrial Zinc Oxide Nanoparticles in a Carbonate-Rich River Water[END_REF][START_REF] Gelabert | Uncoated and Coated ZnO Nanoparticle Life Cycle in Synthetic Seawater[END_REF] For HNO3 2%, NaNO3 0.01M matrices and Seine river water sample, no further dilution was needed prior the HR-ICP-MS analysis. For synthetic seawater and estuarine water samples, 50-fold dilutions were performed for best instrument performance and stability. [START_REF] Mccurdy | Improving ICP-MS Analysis of Samples Containing High Levels of Total Dissolved Solids[END_REF]

Quantitative analysis by ICP-MS

External standard solutions containing 1, 5, 10, 100, 500, 1000 and 5000 ppt of total Cd, Se and Zn were prepared in HNO3 2 %. During the whole analysis, a solution containing 5 ppb of rhodium ( 103 Rh) prepared in HNO3 2% was used as internal standard solution to correct from instrumental drift and mass bias, and connected online to the sample tubing using a T-adaptor. The isotopes of Cd ( 106 Cd, 108 Cd, 110 Cd, 111 Cd, 112 Cd, 113 Cd, 114 Cd and 116 Cd), Zn ( 64 Zn, 66 Zn, 67 Zn, 68 Zn and 70 Zn), and Se ( 74 Se, 76 Se, 77 Se, 78 Se, 80 Se, 82 Se) were analyzed with HR-ICP-MS (ThermoScientific Element II). The isotopes 105 Pd, 115 In, 118 Sn, 60 Ni and 72 Ge were also analyzed to correct possible isobaric interferences. [START_REF] Thomas | A Beginner's Guide to ICP-MS Part XII -A Review of Interferences[END_REF] Each intensity used for data treatment corresponds to the average of 15 blocks of 3 replicate measurements, allowing an internal reproducibility with standard error better than 5%. Within each session, reproducibility of the multi-element reference material (TM-23.4 Lake Ontario water from National Research Council Canada) was checked at the beginning and the end of each analysis sequence and yielded on average 6.6% shift from the certified values. Most of the sub-procedural variation was found to be within the methods stated overall external reproducibility [START_REF] Casadevall | Reproducible Science[END_REF] determined on the three experimental replicates.

Calculation of spiked QDs concentrations

The contrast in isotopic compositions between the multi-spiked QDs and the natural background forms the basis of the quantification of the QDs from HR-ICP-MS measurements. The calculation is an adaptation from Dybowska et al. [START_REF] Dybowska | Synthesis of Isotopically Modified ZnO Nanoparticles and Their Potential as Nanotoxicity Tracers[END_REF] and is detailed in SI.

RESULTS AND DISCUSSION

QDs synthesis and characterization

Prior to the synthesis of multi-spiked QDs, the synthesis protocol was first conducted as a test run using non-isotopically enriched precursors. Green QDs with an emission centered at 540 nm were obtained. Their absorption spectrum features well resolved excitonic peaks and their emission peak has a full width at half maximum (FWHM) of 35 nm, which is in good agreement with the observations of Bae et al. (see SI, Figure S1). When the synthesis was conducted with the isotopically enriched precursors, difficulties were encountered regarding the dissolution of the ZnO precursor. To fully dissolve the powder, it was necessary to heat the metal precursor solution up to 250°C and to maintain it at this temperature for 10 minutes. The resulting multi-spiked QDs also have a green emission centered at 540 nm, however the excitonic peaks are damped and the emission linewidth increases to 40 nm (Figure 1a), indicating that the quality of the nanocrystals has degraded slightly compared to that of the test run QDs. Nevertheless, those properties are still comparable to the QDs described in the original report. The QDs size distribution estimated from a TEM image was 7.9 ± 1.1 nm for n = 250 (Figure 1b andc). An EDXS spectrum is provided in SI (Figure S2) confirming the presence of Zn, Cd, and Se in the QDs.

To allow QDs dissemination in aqueous matrices, the QDs were functionalized by substituting thioglycolic acid (TGA) with the oleic acid ligands stemming from the initial synthesis. TEM images are provided in SI (Figure S2). The isotopic compositions of the multi-spiked QDs regarding Zn, Cd and Se were assessed by HR-ICP-MS and compared to the natural ones (Figure S3). The multi-spiked QDs feature 95.89, 99.11 and 98.95% enrichments in 111 Cd, 68 Zn and 77 Se respectively. These abundances are slightly lower than those certified by ISOFLEX for the starting materials, most probably because of minor contamination by chemicals during the synthesis steps. Nevertheless, the QD modified isotopic composition is in sharp contrast with that of the natural background (12.80%, 18.80%, and 7.64% for 111 Cd, 68 Zn and 77 Se respectively).

Assessment of analytical performances

Recovery plots were built by representing the QDs concentration estimated using Eq. S8 (hereafter referred to as predicted concentration) as a function of the theoretically known concentrations of QDs added in different matrices (reference concentration in the following), as exemplified in the case of the Seine matrix and on the basis of the 68 Zn tracer in Figure 2. The slope of the best linear regression model leads to the recovery rate of the analytical method, namely the fraction of detected QDs amongst the QDs actually present in the sample (which is related to the bias by 𝐵𝑖𝑎𝑠 = (1 -𝑅𝑒𝑐𝑜𝑣𝑒𝑟𝑦) × 100). The precision on the predicted QD concentration was estimated from the Root Mean Square Error of Prediction (RMSEP, defined in Eq. 1) of the regression model. The limit of quantification was chosen as the concentration for which the risk of reporting a false positive or a false negative falls below 0.1%. Briefly, for a set of samples with reference concentration C0, C1, C2, …, Cn where C0 corresponds to the "blank" samples, i.e. to pure matrix samples, the QD-LOQ was practically determined as follows. A t-test was performed to compare the mean value of the predicted concentrations for the blank samples with that of the predicted concentrations for the samples having the reference concentration C1. If the t-test concluded that the two means were significantly different at the 99.9% confidence level, then C1 was designated as the QD-LOQ. If not, then the t-test was conducted using the samples having C2 concentration. The process was repeated with increasing Ci concentrations until the calculated t value exceeded the critical t-value at the 99.9% confidence level (Figure 3). Since our set of QD spiked samples covers several decades, there are only few experimental points in the vicinity of the QD-LOQ. The QD-LOQ determined from the series of t-test conducted on the experimental data then corresponds to an overestimation of the true QD-LOQ. Therefore, a second series of t-tests were conducted using the experimental blank samples and the model predicted values at intermediate concentrations (with 𝐶 𝑖,𝑝𝑟𝑒𝑑 ̂ as the mean and RMSEP as the standard deviation). This estimation is also conservative since it assigns overestimated standard deviations to points at low reference concentrations as the RMSEP is calculated from data covering several decades. The lowest of the two estimates of QD-LOQ was therefore retained.

𝑅𝑀𝑆𝐸𝑃 = √ ∑(𝐶 𝑝𝑟𝑒𝑑,𝑖 -𝐶 ̂𝑝𝑟𝑒𝑑,𝑖 ) 2

𝑛

where n is the number of samples. Eq. 1 S3). The t values for different QDs concentrations in Cd, Zn and Se calculated in all five matrices are also provided in SI (Table S5 to S7).

Analytical performances: QD-LOQ and precision

Figure 4a displays the observed recovery rates by element. The recovery rates for Cd and Zn are excellent at 99 ± 14 % and 97 ± 13 % on average for all investigated matrices. However, the recovery rate for Se is poor at 19 ± 8 %, indicating that the method lacks sensitivity when the 77 Se tracer is used. This is related to the instrumental resolutions at which the three different elements were measured. Briefly, Cd isotopes were measured in low resolution (LR) since this element is not strongly impacted by polyatomic or isobaric interferences. Zn isotopes had to be measured in middle resolution (MR) to solve polyatomic interference issues (e.g. [START_REF] Thomas | A Beginner's Guide to ICP-MS Part XII -A Review of Interferences[END_REF] Ca 16 O + , 32 S 16 O 18 O + , [START_REF] Gottschalk | Modeling Flows and Concentrations of Nine Engineered Nanomaterials in the Danish Environment[END_REF] Ar 14 N2 + ), resulting in counts numbers about 10% of those typically measured in LR. In the case of Se, it was necessary to perform the measurements in high resolution (HR) to overcome polyatomic interferences mostly caused by argon (Ar), e.g. [START_REF] Khan | Stable Isotope Tracer To Determine Uptake and Efflux Dynamics of ZnO Nano-and Bulk Particles and Dissolved Zn to an Estuarine Snail[END_REF] Ar 40 Ar 1 H + , 40Ar2 + , due to the use of Ar as the plasma gas. The intensity signal measured is then around 3% of the total signal measured in LR, which is related to the resolution slit width defining the mass resolution, R (RLR = 300, RMR = 4000, and RHR = 10000). An ANOVA calculation on a model describing the recovery rate as a linear function of the ICP resolution confirmed that the resolution had a significant impact at the 99.9 % confidence level (Tables S9 andS10, model M1). Count numbers were furthered lowered by the fact that the QD concentrations in term of Se were about 2 and 6 times lower on average than that of Cd and Zn respectively. The unsatisfactory results obtained in the case of Se highlight the limits of the spiking method for tracking NPs: for elements subjected to strong polyatomic interferences, it will not be possible to access spike concentrations below the ppb level. However, some specific technical solutions such as hydride generation [START_REF] Far | Determination of the Selenium Isotopic Compositions in Se-Rich Yeast by Hydride Generation-Inductively Coupled Plasma Multicollector Mass Spectrometry[END_REF] could allow overcoming this limitation. Figure 4b and Table 1 display the QD-LOQs of the multi-spiked QDs grouped by tracer element.

In oversimplified media (HNO3 2% and NaNO3 0.01N) where the background levels in Zn, Cd, and Se are below the HR-ICP-MS LOQ (30, 3, and 62 ppt for Zn, Cd and Se, respectively), the QD-LOQ of Zn, Cd, and Se were 10, 0.3, and 6 ppt, respectively, and in NaNO3 0.01N were 7 and 2 ppt for Zn and Cd, respectively. In contrast, in complex matrices such as Seine river water, the Unsurprisingly in light of the poor recovery rates observed for Se, QD-LOQSe are one order of magnitude higher on average than QD-LOQZn and QD-LOQCd. An ANOVA calculation confirmed that the ICP resolution significantly affects the QD-LOQ at the 99% confidence level (Tables S9 andS11, model M2). In the following, the QD-LOQs according to Se will not be further discussed.

For Cd and Zn tracers, QD-LOQs have mean values of 29 and 70 ppt. However, the QD-LOQs in complex matrices (Seine, sea and estuarine waters) are 37 and 11 times those observed in simple matrices (HNO3 and NaNO3) for Cd and Zn respectively. Despite this one-order-of-magnitude increase, the multispiked QDs can be detected in realistic surface water samples in concentrations as low as 41 ppt in Cd and 95 ppt in Zn on average, which are about 3 to 6 order of magnitudes lower than the typical concentrations used in ENPs dissemination and toxicity studies (ppb-ppm level). [START_REF] Khan | Stable Isotope Tracer To Determine Uptake and Efflux Dynamics of ZnO Nano-and Bulk Particles and Dissolved Zn to an Estuarine Snail[END_REF][START_REF] Tang | The Influence of Humic Acid on the Toxicity of Nano-ZnO and Zn2+ to the Anabaena Sp[END_REF][START_REF] Jackson | Toxicity, and Bioaccumulation of Quantum Dot Nanoparticles to the Amphipod Leptocheirus Plumulosus[END_REF][START_REF] Xiao | Sedimentation, Dissolution, and Bioavailability of Quantum Dots in Estuarine Systems[END_REF] These results demonstrate the significant added value of the isotopic labelling technique in order to model the environmental fate of ENPs in conditions mimicking as best as possible the natural media.

The increase of the QD-LOQs in complex matrices compared to the ones in simplified media could be explained by three possible arguments. First, simplified media contain virtually zero geochemical background contribution of the spike elements. Indeed, the concentrations in Cd and Zn in the blank samples in the case of HNO3 and NaNO3 were below the LOQs for their respective isotopes. When moving from these simple media to complex matrices, geochemical background Cd and Zn concentrations increase up to 75 ppt (in sea water) and 3.7 ppb (in Seine water), that is 11 and 3.5 times more than the upper limit of the QD concentration ranges that were investigated in this study. Therefore, the characteristic isotopic fingerprint of the multispiked QDs gets diluted into a large geochemical contribution having the natural isotopic pattern of abundances. This effect will be looked into using the geochemical background concentrations (GBC) of the tracer element in each sample.

Second, complex matrices contain a high concentration of dissolved salts. These can affect intensity readings compared to readings that would have been observed at equal tracer concentration in HNO3, which is the matrix in which the 𝐼( 𝑋 𝐴 ) 𝑣𝑠. 𝐶 𝐴 calibration plots were acquired, in a phenomenon known as matrix effect [START_REF] Garrels | Minerals, and Equilibria[END_REF] which will be investigated using the ionic strength in the solutions analyzed in the instrument as metric.

Finally, the sea water and estuarine water samples contain high dissolved salt concentrations (Table S2, total dissolved solids TDSSW = 40.8 g/L, I = 0.69M) that may damage the instrument and drastically decrease its sensitivity. These samples had to be diluted prior to ICP-MS analysis, to reach at most the maximum recommended TDS values for ICP-MS analysis (2 g/L) [START_REF] Mccurdy | Improving ICP-MS Analysis of Samples Containing High Levels of Total Dissolved Solids[END_REF][START_REF] Fischer | Thermo Fischer Scientific, Application Note 30003, Determination Trace Elements Clinical Samples High-Resolution-ICP-MS[END_REF] resulting in lower tracer concentrations and therefore to lower count numbers. This last effect will be examined using the dilution factor applied to each sample.

On Figure 5, the QD-LOQs according to Cd and Zn tracers were plotted along with the geochemical background concentrations for each matrix and the ionic strength (after dilution if any was required), with an indication of the dilution factor that was applied before ICP-MS analysis.

First, the ionic strength of the solutions analyzed in ICP-MS appears constant throughout the various matrices because the acidification at 2% HNO3 prior to analysis levels it in all analyzed samples. Therefore, matrix effects cannot account for the increase in QD-LOQs observed when moving from simple media to complex aquatic matrices. Second, for both Cd and Zn, the lowest QD-LOQs are the one determined in matrices that did not necessitate dilution prior analysis, so the dilution factor seems to have a significant impact on the QD-LOQ. Last, when Cd is used as a tracer, the QD-LOQs seem to strongly correlate with the GBC. To confirm these graphical findings, ANOVA calculations were conducted. When attempting to model the whole set of QD-LOQs for Cd and Zn, it was found that only the dilution factor and GBC were significant at the 99% confidence level (model M3, Table S12). The QD-LOQs based on Zn were found to depend only on the dilution factor (model M4, Table S13, Figure S25), while those based on Cd had a strong and exclusive correlation with the GBC (model M5, Table S14, Figure S26). These results are in line with the instrumental constraints pertaining to the Zn and Cd detection. As Zn is analyzed in MR for which signal intensity is on the order of 10% of that of LR measurements, it is expected to be more strongly impacted by further shrinking of the count numbers upon dilution than Cd. On the other hand, the LR measurements in the case of Cd have a smaller discrimination power between isotopes: 110 Cd and 111 Cd are less distinct in LR than 66 Zn and 68 Zn in MR, all the more since the latter are separated by two units of mass number. In the presence of increasing GBC of Cd with natural abundance, the 111 Cd/ 110 Cd ratio is damped, which may result in impaired estimation of the 111 Cd contribution. Therefore, the QD-LOQs can be affected by the GBC in the matrices, dilution factor, and analysis resolution. Hence, the relevance of the isotopic spiking method will depend on the element and the matrix targeted in the study. These factors should be considered, for example when choosing the concentration of spiked QDs (or spiked Zn/Cd/Se-containing ENPs) to be used in studies related to ENPs fate and behavior.

To summarize, the limits of quantifications of isotopically labelled CdSe/ZnS quantum dots (QD-LOQs) in natural aquatic matrices were accurately determined by combining HR-ICP-MS and chemometric analysis. QD-LOQs were found varying between 3.4 and 2500 ppt according to the element and the aquatic matrix, which is within the range of ENPs concentrations that are expected in natural surface waters (e.g. 2000, 170, and 100, and <10 ppt for TiO2, ZnO, Ag, and QDs NPs respectively). [START_REF] Gottschalk | Modeled Environmental Concentrations of Engineered Nanomaterials (TiO2, ZnO, Ag, CNT, Fullerenes) for Different Regions[END_REF][START_REF] Gottschalk | Modeling Flows and Concentrations of Nine Engineered Nanomaterials in the Danish Environment[END_REF][START_REF] Piccinno | Industrial Production Quantities and Uses of Ten Engineered Nanomaterials in Europe and the World[END_REF][START_REF] Gottschalk | Environmental Concentrations of Engineered Nanomaterials: Review of Modeling and Analytical Studies[END_REF][START_REF] Gottschalk | Engineered Nanomaterials in Rivers -Exposure Scenarios for Switzerland at High Spatial and Temporal Resolution[END_REF][START_REF] Tiede | Considerations for Environmental Fate and Ecotoxicity Testing to Support Environmental Risk Assessments for Engineered Nanoparticles[END_REF][START_REF] Dumont | Nano Silver and Nano Zinc-Oxide in Surface Waters -Exposure Estimation for Europe at High Spatial and Temporal Resolution[END_REF] Furthermore, our results indicate that the use of isotopic labelling of ENPs coupled with HR-ICP-MS allows to work at ENPs concentrations about 3 to 6 orders of magnitude lower than the typical concentrations used in numerous ENPs fate and toxicity studies (e.g. ZnO at 100 ppb-163 ppm, QDs at 60-3600 ppb). [START_REF] Sivry | Behavior and Fate of Industrial Zinc Oxide Nanoparticles in a Carbonate-Rich River Water[END_REF][START_REF] Gelabert | Uncoated and Coated ZnO Nanoparticle Life Cycle in Synthetic Seawater[END_REF][START_REF] Rocha | The Fate of Polyol-Made ZnO and CdS Nanoparticles in Seine River Water (Paris, France)[END_REF][START_REF] Tang | The Influence of Humic Acid on the Toxicity of Nano-ZnO and Zn2+ to the Anabaena Sp[END_REF][START_REF] Jackson | Toxicity, and Bioaccumulation of Quantum Dot Nanoparticles to the Amphipod Leptocheirus Plumulosus[END_REF][START_REF] Xiao | Sedimentation, Dissolution, and Bioavailability of Quantum Dots in Estuarine Systems[END_REF] Finally, this method does not require the commonly employed feature (e.g. optical emission) for fluorescent ENPs such as QDs, which photoluminescent properties can easily be quenched. The QD-LOQs calculated in the present study are up to 2 orders of magnitude more sensitive than methods based on QDs photoluminescence measurement. 59

Perspective: Relative QD-LOQ

Remarkably, the order of magnitude of relative QD-LOQ (or QD-RLOQ), expressed as the ratio of the QD-LOQ to the sum of QD-LOQ and the total natural element concentration already present in the matrix (Eq. 2) seems to be characteristic of a given element (Table 1). This variability could then be used to draw estimations of the QD-LOQs in matrices not investigated in the present study, provided that the GBC of that new matrix is known. The knowledge of an expected order of magnitude for the QD-LOQ enables to choose the lowest, and therefore closest to the natural media, concentration of QDs to work with in model dissemination experiments, so as to be able to harvest quantitative data by HR-ICP-MS from said experiments. We recommend though to choose such working QD concentration bearing in mind that due to aggregation phenomena, not all disseminated QDs will actually dissolve.

  The final TGA-coated QDs stock solution in pH 10 water contains 101.1 ± 1.0 ppm of Zn, 31.8 ± 0.3 ppm of Cd, 19.2 ± 0.3 ppm of Se, and 404 ± 41 ppm of S by ICP-OES.

Figure 1 .

 1 Figure 1. a) UV-vis absorption spectrum (blue line) of the multi-spiked quantum dots and

Figure 2 .

 2 Figure 2. 𝐶 𝑝𝑟𝑒𝑑 𝑣𝑠. 𝐶 𝑟𝑒𝑓 recovery plots for estimations of QDs concentration based on the 68 Zn

Figure 3 .

 3 Figure 3. Overview of methodology applied to estimate the QD-LOQ from the recovery plot or its

Figure 4 .

 4 Figure 4. Recovery rates grouped by tracer element, for all matrices confounded (a) and QD-LOQs

2 (

 2 QD-LOQZn increases to 39 ppt. In estuarine water 1 (Seine/SW 75:25), estuarine water Seine/SW 50:50), estuarine water 3 (Seine/SW 25:75), and SW, the QD-LOQZn increases to 139, 118, 64, and 112 ppt, respectively. The QD-LOQ in Seine, estuarine water 1, 2, 3, and SW, were 3.4, 22, 39, 76, and 62 ppt, respectively for Cd and 132, 1044, 2534, 2003, and 1689 ppt,

Figure 5 .

 5 Figure 5. Limits of quantification of multi-spiked QDs (QD-LOQs, dots, in ppt), geochemical

  to accurately assess the efficiency of the spiking method by considering the GBC in the matrix. Globally, we propose that the RLOQ values defined in the present study can be further used to determine the concentration of Zn/Cd/Se-containing ENPs to be used in any experiment related to their fate/behavior/toxicity by using isotopic labelling technique. However, one should consider a slightly higher ENPs concentration when studying their dissolution since the dissolved ENPs concentration is initially unknown. For instance, if Zn from Zn-containing ENPs is expected to dissolve at most 50%, the ENPs concentration used should be at least 2 times the QD-LOQ. Future studies related to ENPs fate, behavior, and toxicity in aquatic environments should be, as much as possible, carried out at environmentally relevant concentrations through the use of isotopically labelled ENPs, on the basis of the QD-LOQs estimated in the present study. The use of relevant concentrations in future experimental work should result in a better and accurate understanding on the ENPs fate, behavior, and toxicity.

Table 1 .

 1 GBC, QD-LOQ, precision (RMSEP), and RLOQ in different matrices. The conventional HR-ICP-MS LOQ are: Zn 30 ppt, Cd 3 ppt, Se 60 ppt. n.a.: not available.

			NaNO3		Seine/SW	Seine/SW	Seine/SW
		HNO3 2%		Seine				SW
			10 -2 M		75:25	50:50	25:75
	GBC (ppt)	< LOQZn	< LOQZn	3718 ± 186	3416 ± 171	2169 ± 108	1493 ± 75	1345 ± 67
	QD-LOQ (ppt)	10	7	39	139	118	64	112
	Zn
	Precision (ppt)	0.8	1	8	27	42	38	23
	QD-RLOQ	-	-	1.0%	3.9%	5.2%	4.1%	7.7%
	GBC (ppt)	< LOQCd	< LOQCd	8.4 ± 0.4	23 ± 1.2	42 ± 2.1	81 ± 4.0	75 ± 3.8
	QD-LOQ (ppt)	0.31	1.9	3.4	22	39	76	62
	Cd
	Precision (ppt)	0.11	0.30	0.78	7.5	8.9	14	10
	QD-RLOQ	-	-	29%	49%	48%	48%	45%
	GBC (ppt)	< LOQSe	n.a.	1069 ± 53	933 ± 47	649 ± 33	617 ± 31	683 ± 34
	QD-LOQ (ppt)	6	n.a.	132	1044	2534	2003	1689
	Se
	Precision (ppt)	0.4	n.a.	5	25	64	36	36
	QD-RLOQ	-	-	11%	53%	80%	77%	71%

Funding Sources

This research project (nanospike.fr) was fully supported by the French Agency for Food, Environmental and Occupational Health & Safety (ANSES) under the convention N° EST-2013/1/264. Part of this work was supported by the IPGP multidisciplinary program PARI and by Paris-IdF region SESAME Grant n°12015908.

For Table of Contents Only

ASSOCIATED CONTENT

Supporting Information

Compositions of aquatic matrices (Tables S1 andS2), Acid digestion protocol, Calculations of spiked QDs concentrations for Zn, Cd, and Se, Absorption and emission spectra of the test run QDs (Figure S1), QDs characterization images (Figure S2), Isotopic composition of multispiked QDs (Figure S3), Daily determination of the conventional HR-ICP-MS limit of quantification (LOQ) and its standard error (Figure S4), HR-ICP-MS calibration and spiked QDs concentration recovery plots (Figures S5 to S24), bias of the recovery plots (Table S3), t table (Table S4), calculated statistic t values (Table S5 to S7), ANOVA test tables (Table S8 to S14) and correlation figures (Figures S25 andS26).

This material is available free of charge via the Internet at http://pubs.acs.org

AUTHOR INFORMATION

Corresponding author

*Email: sivry@ipgp.fr

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. † ‡ ∆ These authors contributed equally.

Notes

The authors declare no competing financial interest. ABBREVIATIONS ENPs, engineered nanoparticles; FWHM, full width at half maximum; GBC, geochemical background concentrations; QDs, quantum dots, QD-LOQ, limit of quantification of spiked QDs according to the element labelled and to the matrix composition; QD-RLOQ, relative QD-LOQ.