Bérengère Piron
email: berengere.piron@chu-nimes.fr

D Broggio

M Bardiès

C Barrau

P O Kotzki

V Boudousq

Dosimetric methodology for 131 I therapy for benign thyroid diseases Méthodologie pour la dosimétrie des traitements à l'iode 131 des maladies bénignes de la thyroïde

Le traitement à l'iode 131 fait partie des traitements de référence pour les maladies bénignes de la thyroïde. Habituellement, la dose prescrite est unique pour une même maladie. Inspirée de la radiothérapie externe, l'émergence de la dosimétrie personnalisée pré-thérapeutique dans les services de médecine nucléaire permet de s'adapter à la maladie et à la physiologie du patient ; plus particulière à sa cinétique de distribution de l'iode. En personnalisant l'activité prescrite, la dosimétrie permet de se conformer au principe ALARA en radiothérapie métabolique tout en préservant l'objectif thérapeutique. La dosimétrie post-traitement permet quant à elle de valider la méthodologie et, en relevant la dose réellement délivrée aux organes avoisinant le volume cible, de contribuer à la compréhension de la relation dose-effet. Après un rappel du formalisme du MIRD sur lequel s'appuie le calcul de dose, ce document recense la démarche à suivre en dosimétrie thyroïdienne. Définition de la sensibilité, calcul des facteurs S, estimation de l'activité cumulée et des temps de résidence puis calcul de la dose sont les différentes étapes présentées. La nécessité d'une bascule de la prescription en dose fixe à la prescription personnalisée semble aujourd'hui être une évidence. De nombreuses études en cours ainsi que l'émergence de nouveaux logiciels compilant l'ensemble des informations indispensables pour sa mise en oeuvre permettent aux services de médecine nucléaire de se projeter plus facilement dans la démarche.

Dosimétrie personnalisée-Traitement à l'iode 131-Maladie bénigne de la thyroïde

Abstract

Treatment with 131 I is one of the standard treatments for benign thyroid disease. In most cases, the prescribed dose is specific for the same disease. Inspired by external radiotherapy, the emergence of personalized pre-therapeutic dosimetry in nuclear medicine departments makes it possible to adapt to the patient's diseases and physiology; more specific to its kinetics of iodine distribution. By personalizing the prescribed activity, dosimetry makes it possible to comply with the ALARA principle in metabolic radiotherapy while at the same time preserving the therapeutic objective. Post-treatment dosimetry permits to validate the methodology and, by recording the dose actually delivered to the organs surrounding the target volume, to contribute to the understanding of the dose-effect relationship. A rereading of the MIRD formalism guidelines is important as this document lists the procedure to be followed in thyroid dosimetry. Defining the sensitivity, calculating the S factors, estimating the cumulative activity and residence times and then calculating the dose are the different steps presented. The need to switch from fixed dose prescription to personalized prescription seems to be evident. Several studies in progress as well as the emergence of new software compiling all the information essential for its implementation will permit nuclear medicine departments to involve themselves more easily into the process.

Personalized dosimetry-Radiodine therapy -Benign thyroid disease

Introduction

Treatment of hyperthyroidism including Graves' disease, toxic multinodular goiter and toxic solitary nodule with 131 I is routinely performed in nuclear medicine departments. The activity of administration can be determined using two approaches, empiric and dosimetry-based.

The current trend in France [START_REF] Bernard | Radioiodine therapy in benign thyroid disorders. Evaluation of French nuclear medicine practices[END_REF] and in many European countries is the administration of fixed activities, "one-size-fits all" or modulated by the clinical status of the patient (empiric approach). However, a more personalized approach, dosimetry-based, can also be proposed.

This complies with EURATOM directive 2013/59 [START_REF] Teunen | The European Directive on health protection of individuals against the dangers of ionising radiation in relation to medical exposures (97/43/EURATOM)[END_REF] from the Council of the European Union, which states that for all medical exposure of patients for radiotherapeutic purposes, exposures of target volumes shall be individually planned (article 56 Optimization).

Implementing patient specific dosimetry -and adapting the administered activity on a patientspecific base allows implementing the "ALARA" principle (As Low As Reasonably Achievable), while preserving the therapeutic objective (…consistent with the intended radiotherapeutic purpose of the exposure).

The concept of treatment planning is very well accepted in external beam radiotherapy (EBRT). It is tempting to establish a parallel with targeted radionuclide therapy (TRT), even though radiation delivery is very different -which may impact the radiobiology and therefore the clinical outcome of the treatment.

In EBRT, the process is to simulate the treatment by calculating a dose distribution map, verify it and then assess how the treatment plan is implemented during the therapy. This means comparing post-treatment dosimetry with pre-treatment dosimetry.

In TRT, the aims associated with dosimetry can be different, as pre-therapeutic and posttherapeutic dosimetry can be implemented with different purposes:

-Pre-therapeutic dosimetry aims at tailoring the treatment according to a dosimetric index. The concept of treatment planning in TRT relies on the assumption that pharmacokinetics obtained from a pretherapeutic procedure can predict that of the actual treatment. In addition, administering a given activity based on a dosimetric parameter (the absorbed dose to the clinical target, or that to a critical organ) relies on the knowledge of the absorbed dose -effect relationship (as is the case in EBRT).

-Post-therapeutic dosimetry can be implemented to document the radiotherapeutic procedure, even when routine activities are administered. The assessment of absorbed doses delivered after activity administration allow understanding the clinical outcome.

In addition, like in EBRT, post-therapeutic dosimetry can be implemented to verify the delivered treatment after pre-therapeutic dosimetry.

For the treatment of benign thyroid disease, several journals reported an absorbed doserelated outcome after radioiodine treatment [3; 4] and a correlation has been identified between hypothyroidism and absorbed dose, while the irradiation of non-target tissues has also emerged as a factor that needs careful consideration [START_REF] Canzi | Verification of the agreement of two dosimetric methods with radioiodine therapy in hyperthyroid patients[END_REF].

In consequence, for the benefit of patients it might be suitable to tailor the administrated activity according to their biokinetics and thus avoid under-or over treatment. That is why developments are oriented on personalized dosimetry to improve the quality and outcome of radionuclide therapy [6; 7]. In this context, personalized treatment should be particularly encouraged for children who have a higher risk than adults to develop radiation-induced cancer.

By exposing an overview of dosimetry approaches, the aim of this document is to provide information to help implementing dosimetry procedures in nuclear medicine departments.

Dosimetric Methodology

We will present the basics of radiopharmaceutical dosimetry, and then see how they can be applied to the specific situation of the therapy of thyroid benign disease with 131 I as presented in the European Association of Nuclear Medicine (EANM) guidelines [8; 9].

Radioisotopes for dosimetry

For pre-therapeutic dosimetry, a low activity of 131 I can be used [START_REF] Skanjeti | A simple and accurate dosimetry protocol to estimate activity for hyperthyroidism treatment[END_REF]. Alternatively, the distribution of 131 I can be estimated indirectly using a surrogate isotope like 123 I, with similar biological and chemical characteristics, but better suited to scintigraphic imaging (gamma emission of 159 keV and physical half-life of 13.2 hours) [START_REF] Li | Quantitative Imaging for Targeted Radionuclide Therapy Dosimetry -Technical Review[END_REF].

Recent studies proposed using 124 I with positron emission tomography (PET) for pretherapeutic dosimetry [11; 12] and compared 124 I and 131 I uptake in patients. 124 I is 511 keV gamma emitter with a half-life of 4.18 days.

For post-therapeutic dosimetry, 131 I administrated for the treatment is used. Iodine 131 is a beta-emitting radionuclide with a physical half-life of 8.02 days. The maximum energy of the main beta emission is 610 keV (average beta energy of 192 keV), which corresponds to an average range of 0.4 mm in soft tissues thereby assessing localized irradiation as is desirable in TRT. The principal gamma ray of 364 keV makes it possible (albeit not optimal) to visualize and quantify activity in the patient [START_REF] Eckerman | ICRP Publication 107. Nuclear decay data for dosimetric calculations[END_REF].

Phantom and calibration measurements

The quantification of activity present in patient body requires implementing calibration procedures. Calibration should be made for each isotope used in clinical practice.

Series of phantom measurements including known activities are performed and used for quantitative studies. Aside from the determination of camera sensitivity, acquisitions performed in phantoms that mimic the clinical situation allow implementing attenuation, scatter or partial volume effect corrections.

Basically, the calibration should be performed using a phantom with a geometry and a composition as close as possible to that of the human neck, to replicate radiation absorption and scatter in the patient's thyroid [START_REF] Fujita | Investigation of post-therapeutic image-based thyroid dosimetry using quantitative SPECT/CT, iodine biokinetics, and the MIRD's voxel S values in Graves' disease[END_REF].

Simple thyroid phantoms (such as simple cylinders) have been routinely used in clinical practice, but present limitations due to their simplistic geometry [START_REF] Vahjen | Thyroid uptake neck phantoms are not created equal[END_REF]. More recently, some teams have proposed more realistic and low cost thyroid phantoms based on 3D printing [START_REF] Alssabbagha | Evaluation of 3D printing materials for fabrication of a novel multi-functional 3D thyroid phantom for medical dosimetry and image quality[END_REF][START_REF] Beaumont | Development and test of sets of 3D printed age-specific thyroid phantoms for (131)I measurements[END_REF][START_REF] Kuhnel | 3D printing of fillable individual thyroid replicas based on nuclear medicine DICOM data used as phantoms for gamma probe calibration[END_REF]. These efforts may lead to improved calibrations of the imaging devices or thyroid counters.

The tracer activity used for dosimetry should be measured in the phantom prior to the administration. Image/counting acquisitions should be performed with a phantom placed at the same distance to the camera than the patent's neck. The resulting count rate value represents a 100 % uptake, and the count rate divided by the activity should be used as a constancy check for the sensitivity of the device [START_REF] Hanscheid | EANM Dosimetry Committee series on standard operational procedures for pre-therapeutic dosimetry II. Dosimetry prior to radioiodine therapy of benign thyroid diseases[END_REF].

The system volume sensitivity, which is the conversion factor used for calculating radioactivity from SPECT counts, can be obtained according to the method described by Frey et al. [START_REF] Frey | Accuracy and precision of radioactivity quantification in nuclear medicine images[END_REF].

Verifying the constancy of detector sensitivity for a reproducible geometry is recommended and its periodicity should be defined in each nuclear medicine department as a function of activity. Ideally, the sensitivity should be evaluated by measuring the activity to be administered before each administration, in both by the measuring device and by an activimeter [START_REF] Hanscheid | EANM Dosimetry Committee series on standard operational procedures for pre-therapeutic dosimetry II. Dosimetry prior to radioiodine therapy of benign thyroid diseases[END_REF].

SPECT/CT: acquisition and correction

SPECT/CT is used to perform the uptake measurements. With 123 I, a low energy collimator with a high resolution is used for acquisition. For iodine 131, possibly used for pre and posttherapeutic dosimetry, a high energy collimator is used for scintigraphy to reduce septal penetration. If available, a camera with a thick NaI(Tl) crystal should be selected to increase detection sensitivity and count rate. Such cameras can be expected to record enough counts, about 50 counts per second per mega Becquerel for 131 I in a 15 % window centered on the main emission peak at 364 keV [START_REF] Hanscheid | EANM Dosimetry Committee series on standard operational procedures for pre-therapeutic dosimetry II. Dosimetry prior to radioiodine therapy of benign thyroid diseases[END_REF]. The distance between camera and patient should be as small as possible to improve spatial resolution.

To improve image quality and allow activity quantification, different corrections must be made like a background correction in planar imaging, attenuation correction using CT in hybrid SPECT/CT systems, scatter correction, partial volume effect correction and dead time [20; 21].

Periodic checks must be carried out on the SPECT/CT to ensure the constancy of imaging performances.

Thyroid mass assessment

The determination of thyroid volume is essential, as the absorbed dose is inversely proportional to the mass of the volume of interest. Thyroid volume is most often assessed via 2D or 3D ultrasonography (US) [START_REF] Licht | 3D ultrasonography is as accurate as low-dose CT in thyroid volumetry[END_REF][START_REF] Nygaard | Thyroid volume measured by ultrasonography and CT[END_REF][START_REF] Reinartz | Thyroid volume measurement in patients prior to radioiodine therapy: comparison between three-dimensional magnetic resonance imaging and ultrasonography[END_REF]. Magnetic resonance imaging or computed tomography [START_REF] Freesmeyer | Multimodal evaluation of 2-D and 3-D ultrasound, computed tomography and magnetic resonance imaging in measurements of the thyroid volume using universally applicable cross-sectional imaging software: a phantom study[END_REF][START_REF] Van Isselt | Comparison of methods for thyroid volume estimation in patients with Graves' disease[END_REF][START_REF] Lee | Semiautomated thyroid volumetry using 3D CT: prospective comparison with measurements obtained using 2D ultrasound, 2D CT, and water displacement method of specimen[END_REF] have also been proposed.

Ultrasound measurements were compared with reference MRI measurements and found to correlate well, provided that the ellipsoid formula [START_REF] Hanscheid | EANM Dosimetry Committee series on standard operational procedures for pre-therapeutic dosimetry II. Dosimetry prior to radioiodine therapy of benign thyroid diseases[END_REF] used in ultrasonography is corrected.

When comparing US measured volumes with surgically removed volume, mean differences of 13% and 22% were found by Hussy et al. [START_REF] Hussy | Determination of the thyroid volume by ultrasonography -Comparison with surgical facts[END_REF] and Miccoli et al. [START_REF] Miccoli | Ultrasonography estimated thyroid volume: a prospective study about its reliability[END_REF], respectively.

Comparing 2D and 3D US measurements, [START_REF] Andermann | Intra-and interobserver variability of thyroid volume measurements in healthy adults by 2D versus 3D ultrasound[END_REF] found that 2D overestimated the volume by 17%. Even if the overall accuracy of US might be acceptable for absorbed dose assessment [START_REF] Lucas | Use of thyroid ultrasound volume in calculating radioactive iodine dose in hyperthyroidism[END_REF], the experience of observers is an important factor affecting the precision of measurements [23; 30; 32].

MIRD formalism

According to the MIRD formalism, consistent with the ICRP formalism, the absorbed dose delivered to a given target k is the sum of the contributions of every source h present in the patient:

(1).

is the cumulated activity in the source (or time integrated activity, which means residence time) and represents the number of nuclear disintegrations occurring in the source h (in MBq.s or equivalent).

is called the "S-factor" and gives the absorbed dose delivered in target k per nuclear disintegration in the source h. It is more convenient to express S in Gy.MBq-1.days-1.

Thyroid S-factors

For thyroid dosimetry following 131 I administration, the previous equation can be simplified since (i) most of the up taken activity stays in the thyroid, and (ii) the fraction of photon energy emitted in the thyroid and absorbed in the thyroid (self-absorbed) is extremely small, whereas (iii) most of the electrons/beta energy emitted in the thyroid is absorbed in the thyroid.

In that context, the S-factor for 131 I in Gy.MBq -1 .day -1 can be given by:

(2).

For a thyroid weight less than 90 g this simple relation gives error less than 5% [START_REF] Hanscheid | EANM Dosimetry Committee series on standard operational procedures for pre-therapeutic dosimetry II. Dosimetry prior to radioiodine therapy of benign thyroid diseases[END_REF]. A more precise expression is:

(3)
Alternative expressions for the S-factors are provided in [34; 35] but they do not represent significant change to those presented here.

Biokinetic model for the thyroid

To calculate the number of nuclear disintegration in a given organ ( ) one needs a biokinetic model describing the thyroid activity content along time. The biokinetic model adopted by the EANM [START_REF] Hanscheid | EANM Dosimetry Committee series on standard operational procedures for pre-therapeutic dosimetry II. Dosimetry prior to radioiodine therapy of benign thyroid diseases[END_REF] is reproduced in Figure 1.

In this simple model the activity in the thyroid is described by three transfer rates, the blood to thyroid transfer rate (kt), the renal clearance (kr), the thyroid hormone clearance (kh). The physical decay constant is noted kp. For consistency with the previous relations the transfer rates and physical decay constant must be expressed in days -1 . The transfer rates are patient dependent but the physical decay depends on the radio-iodine isotope, the biokinetic model is thus not specific of 131 I.

The biokinetic model enables the calculation of the fractional Radio-Iodine Uptake (RIU) in the thyroid at a given time, i.e. the fraction of the administrated activity (Aa) that is in the thyroid at a time t:

(4)
The advantage of the simple biokinetic model is that RIU(t) can be calculated as follows:

(5)

The parameters kB and kT are defined as follows:

(6a)

.

(6b)

At this stage it can be noticed that the long-term trend of RIU(t) is given by the first time in parenthesis of Eq. 5 and it thus enables to define the effective half-live of radio-iodine in thyroid as follows:

(7)
The cumulated activity is the integral of RIU(t) multiplied by the administrated activity:

(8).
The last term stands for the residence time (or time integrated activity coefficient) in the thyroid. It is the integral of RIU(t) and has units of days.

Residence time assessment from measurements and activity prescription

It is assumed that activity measurements in the thyroid are taken at early (te) and/or late (tl) time after administration of a tracer activity of 131 I.

Once is determined, the activity to administer to obtain the therapeutic absorbed dose can be calculated using Eq. 9 described below. Table 1 gives approximations for assessment.

Pre-therapeutic dosimetry and prescription of administrated activity

To deliver a given therapeutic absorbed dose to the thyroid ( ) one thus needs:

-The S-factor that is easily calculated from the thyroid mass (cf section above);

-The biokinetic parameters describing the thyroid uptake (kt, kT, kB) or an estimate of the resident time ( )

Then the activity required to reach a given absorbed dose is given by:

(9)
Depending on the time (after tracer activity administration) and on the numbers of thyroid uptake measurements different approximation can be given for , as presented in the previous paragraph that summarizes the EANM guidelines.

Table 2 presents numerical examples for the cases described in Table 1. Starting from the thyroid mass the S factor is calculated with Eq. 2. The residence time is calculated from Table 1. The administrated activity required to reach a given therapeutic absorbed dose is obtained from Eq. 9.

Post-therapeutic dosimetry

Even if low activity of 131I is administrated in the pre-therapeutic phase it cannot be ruled out that thyroid stunning might occur, and even if stunning is not induced by the pre-therapy administration the administrated therapy activity might modify the thyroid uptake parameters [36; 37]. Similarly, the time elapsed since anti-thyroid drug withdrawal might induce change in thyroid uptake [START_REF] Osaki | Prediction of late (24-hour) radioactive iodine uptake using early (3-hour) uptake values in Japanese patients with Graves' disease[END_REF][START_REF] Walter | Radioiodine uptake and thyroid hormone levels on or off simultaneous carbimazole medication: a prospective paired comparison[END_REF][START_REF] Zhang | Prediction of thyroidal (131)I effective half-life in patients with Graves' disease[END_REF], thus thyroid uptake during the pre-therapy and therapy phase are not necessarily the same.

Consequently, post-therapy dosimetry is useful to adjust following treatments if the current one fails and to document at best the patient records.

The methods exposed in the previous sections for pre-therapeutic dosimetry remains valid for post-therapeutic dosimetry. Uptake measurements can be obtained to check that the administered activity actually led to the prescribed absorbed dose . A study by Fujita et al. investigated post-therapeutic imaging for dosimetry [START_REF] Fujita | Investigation of post-therapeutic image-based thyroid dosimetry using quantitative SPECT/CT, iodine biokinetics, and the MIRD's voxel S values in Graves' disease[END_REF]. The absorbed dose delivered was calculated based on quantitative SPECT/CT, on the basis of the guidelines proposed by the MIRD committee for voxel dosimetry. Patient SPECT images were acquired 24 h after oral administration to confirm iodine uptake in the thyroid, and at various time intervals to determine time activity curve.

We exposed pre-therapeutic and post-therapeutic implementation of 131 I dosimetry for the treatment of benign thyroid disease. The most important point is the assessment of patientspecific biokinetic parameters, which can be done also with other radio-iodine isotopes [12; 41]. To adapt the method to other radio-iodine isotopes with shorter half-live it would be needed to:

-Administer enough activity so that late measurements are not under detection limits;

-Perform a physical decay correction to transform the RIU to the 131 I equivalent.

Other iodine biokinetic models are available [START_REF] Leggett | A physiological systems model for iodine for use in radiation protection[END_REF]. If such models were used it is not guaranteed that the residence time assessment presented in the EANM guidelines would remain strictly valid. In that situation, an alternate way to assess the residence time would be needed to grant a correct dosimetric prescription.

Perspectives

This document provide an overview of the dosimetric approaches for radiodine treatment of benign thyroid diseases.

The relevance of dosimetry is not questioned in external beam radiotherapy, where treatment planning has been routinely performed for decades. A number of justifications are frequently presented to avoid dosimetry in nuclear medicine therapy [43; 44], like the prohibiting cost, the uncertainties associated with absorbed dose determination [START_REF] Gear | EANM practical guidance on uncertainty analysis for molecular radiotherapy absorbed dose calculations[END_REF] or the difficulty to make evident the absorbed dose-effect relationship [START_REF] Brans | Clinical radionuclide therapy dosimetry: the quest for the "Holy Gray[END_REF].

Studies are necessary to improve the correlations between the administered activity and the clinical outcome and enable better understanding of the absorbed dose-response relationship.

Different groups are working to reach this goal.

As described, both pre-therapeutic and post-therapeutic dosimetry are performed using the MIRD formalism.

Table 1 Three or more uptake measurements Measurements should be at least three and around 4-6 hours, 1-2 days and 5-8 days. Measurements are used to fit RIU(t) and deduce kt, kB, kT from which is assessed. An example is provided in [START_REF] Hanscheid | EANM Dosimetry Committee series on standard operational procedures for pre-therapeutic dosimetry II. Dosimetry prior to radioiodine therapy of benign thyroid diseases[END_REF].

Two

  At the same time, research work and clinical needs have stimulated the emergence of commercial software enabling to directly compute patient-specific, image-based absorbed dose (rate) maps at the voxel level, from anatomic/functional data derived from hybrid imaging technologies. As is current practice in EBRT, absorbed dose volume histograms can be generated from these new software. Biological Equivalent Dose (BED) and a range of dosimetry-related indexes can be obtained, even though it remains to be seen if voxel-based dosimetry can provide a better appraisal of clinical response[54].These softwares may have an FDA/EAM approval or CE marking as medical device, making them a viable option to implement dosimetry in clinical routine. Studies are in progress to identify "dosimetry-based biomarkers of response" for TRT, and the availability of userfriendly software is a further motivation for deploying personalized dosimetry in nuclear medicine departments. 52. Andersson M, Johansson L, Eckerman K, Mattsson S. IDAC-Dose 2.1, an internal dosimetry program for diagnostic nuclear medicine based on the ICRP adult reference voxel phantoms. EJNMMI Res 2017;7:88 53. Chauvin M, Borys D, Botta F et al. OpenDose: open access resources for nuclear medicine dosimetry. J Nucl Med. 2020 10.2967/jnumed.119.240366 54. Chiesa C, Bardies M, Zaidi H. Voxel-based dosimetry is superior to mean absorbed dose approach for establishing dose-effect relationship in targeted radionuclide therapy. Med Phys 2019;46:5403-6

	The concept of S value implicitly refers to model-based dosimetry, where tables of
	precomputed S values are calculated for anthropomorphic dosimetric models. Codes like the
	well-known OLINDA/EXM implements this model-based approach [47; 48]. Interestingly, S
	values can be adapted when the mass of organs/tissues differ from that of the reference model
	[49]. A new generation of voxel-based and more realistic human models was proposed by
	various authors [50] and implemented in more recent software like OLINDA/EXM 2.0 [51]or

IDAC [52]. The recently created OpenDose collaboration aims at providing open access resources (including S values) for nuclear medicine dosimetry [53].

uptake measurements : early and late

	Conditions	approximation
	1≤te≤2 days
	4≤tl≤8 days	If Teff is more than 8 days, it is set to 8 days
	tl-te≥3 days
		One late uptake measurement
	Conditions	approximation
	4≤tl≤8 days
		One early uptake measurement
	Conditions	approximation
	1≤te≤3 days
		Test is the estimated effective half-life.

Table 2

 2

			Therapeutic
	Case	Thyroid mass (g)	absorbed dose	te (days)	tl (days)	RIU(te) RIU(tl)	Aa (MBq)
			(Gy)
	Two uptake measurements	20	300	1.5	6	0.4	0.25	491.6
	Two uptake
	measurements.	10	300	1	4	0.35	0.3	224.8
	Teff>8 days
	One late measurement	20	150		7		0.2	272.5
	One early
	measurement	15	250	2		0.45		299.5
	Test=5.5 days

ACKNOWLEDGMENTS:

The authors would like to thank Joël Greffier from Medical Physics Unit of Nîmes University Hospital for his support.

CONFLICT OF INTEREST

The authors confirm that there is no conflict of interest


MD Vivien Hébert

MD Anne-Bénédicte Duval-Modeste

MD, PhD Pascal Joly

MD Véronique Lemée

MD Lucie Cellier

MD Fabienne Jouen

Benoit Veber

PhD Laurent Drouot

MD, PhD Olivier Boyer

MD, PhD ; Benoit Veber

Lack of association between chilblains outbreak and severe acute respiratory syndrome coronavirus 2: Histologic and serologic findings from a new immunoassay

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Recently, numerous cases of acute chilblains-like lesions have been reported on social networks, from Belgium, France and Italy. Despite weak evidence, in particular the absence of serological studies, the hypothesis that these lesions were potentially related to the Covid-19 infection, as a paucisymptomatic form, rapidly grew [START_REF] Bouaziz | Vascular skin symptoms in COVID-19: a french observational study[END_REF][START_REF] De Masson | Chilblains are a common cutaneous finding during the COVID-19 pandemic: a retrospective nationwide study from France[END_REF][START_REF] Kolivras | Coronavirus (COVID-19) infection-induced chilblains: a case report with histopathological findings[END_REF] . An alert letter was even sent to the French Ministry of Health and to French dermatologists, by the National Union of Dermatologists.

Since 33 patients (14 women, 19 men) with chilblains were referred to our Dermatology Department within one week, we studied the relationship between these lesions and the Covid-19 infection. All patients (mean±SD age 23.4±8.7 years.) presented erythematous and purpuric papules localised on the toes ± fingers (12 patients, 36.4%) (Figure1). Edema was present in 12 (36.4%), and pruritus or burning sensation in 18 cases (54.5%). Median delay between the onset of dermatological features and the first consultation was 8 days (IQR 6.5-18 days). Chilblains were the only clinical manifestation in 23 cases (69.7%); ten patients (30.3%) presented other symptoms which occurred prior to (n=6), concomitantly (n=1) or after skin symptoms (n=3) : asthenia (n=4), cough (n=3), diarrhoea (n=3), fever (n=2), myalgia (n=2), headache (n=1) and odynophagia (n=1). No patient reported contact with Covid-19 infected patients. Three patients recently tested for Covid-19 infection had negative sinus RT-PCR.

Blood cell count was normal in 26 patients. A mild lymphopenia (mean 1.15 ±0.21 G/L) was observed in seven patients. C-reactive protein and erythrocyte sediment rate were negative for all patients. Two patients had positive ANA and three patients had Abs for a type III cryoglobulinemia. Histology performed in five patients showed lymphocytic infiltrate in the superficial dermis around vessels and eccrine glands in all cases, reminiscent of idiopathic chilblains. Direct immunofluorescence showed fibrinogen and C3 deposits on endothelial cells in two cases. IIF assay using the serum from a patient with anti-SARS-CoV-2 IgG Abs, and RT-PCR on lesional skin were negative.

We developed an ALBIA-Spike S1 for detecting anti-SARS-CoV-2 antibodies, that allowed a 96% sensitivity and a 98% specificity (Drouot et al. in preparation). Anti-SARS-CoV-2 IgG Abs were negative in all 33 sera, while 1/33 was positive for IgM at the first and the second consultation 14 days later. No seroconversion was observed (Figure 2). Our findings were then confirmed using the Abbott® SARS-CoV-2 IgG immunoassay performed on the Day 14 sera, without detectable seropositivity.

The present data provide no argument for any link between these chilblains and infection with SARS-CoV-2. Clinical and histological features were those of idiopathic chilblains. RT-PCR and IIF on lesional skin when performed were negative. None of the 33 sera tested contained anti-SARS-CoV-2 IgG Abs, while only one had IgM twice (3%), consistent with the current estimation of a rate of SARS-CoV-2 infection from 5% to 10% in the general population in France (6).

We think that this hypothesis of "Covid-19 related chilblains" could be explained by a cumulation of i) a temporality bias in this early-spring period when the average temperature differences were the widest, ii) a confounding bias related to the young age, since pauci-symptomatic forms of the infection are observed in young people, and iii) a recruitment bias related to the shortening of the dermatology consultation delays due to the quarantine period.

Figures / Tables = 2 I

 2 Figures / Tables= 2


Desaulty Anne-Marie

Perret Sebastien

Maubec Nicolas

Négrel Philippe

Tracking anthropogenic sources in a small catchment using Zn-isotope signatures

Keywords: Heavy metals, Zn isotopes, Contamination, Source tracer, Catchment

Tracking metal pollution in surface water is a major environmental, public-health and economic issue. Knowledge of the behaviour of metals such as zinc (Zn), in stream sediments and water, is a key factor to improving river water quality. Because the isotopic compositions of Zn can be used as an environmental tracer for tracking the sources of manmade materials and its transport into water and soil, we have used Zn isotopes for studying the anthropogenic impact on a watershed. As a case study, we chose a small watershed in the Loire River basin, near Orléans (France). The Egoutier spring issues in a pristine area, but its water is affected a few kilometres downstream by liquid effluents from a wastewater treatment plant (WWTP) and farther down still by diffuse pollution from road traffic. We sampled the liquid effluents as well as water and sediments along an upstream to downstream transect. For liquid samples, we took "grab" samples and integrated samples using passive DGT (Diffusive Gradients in Thin films) samplers. The advantages of DGT are (1) to pre-concentrate in-situ the Zn dissolved in water-avoiding a time-consuming chemical preparation-and (2) to integrate the potential variations of Zn isotopic signature over time.

The mobile fraction of sediments-that released by the leaching in 0.2N HCl-was also investigated. The δ 66/64 Zn values in water and sediments along the upstream/downstream profile clearly showed the anthropogenic input of WWTP effluents in the river, which was still visible several kilometres downstream in water. This dissolved anthropogenic contribution is

Introduction

Zinc (Zn) is a ubiquitous metal present in all soils, plants and biota. It plays a dual role in the environment, being either an essential or a toxic element for organisms depending on its concentration, its speciation in solution and the organism [START_REF] Campbell | Interactions between trace metals and aquatic organisms: a critique of the freeion activity model[END_REF][START_REF] Whitton | Toxicity of heavy metals to freshwater algae: a review[END_REF].

For example, Zn depletion leads to nutrient limitation in the ocean, and Zn excess can cause reproductive, developmental, behavioural and toxic responses in a variety of aquatic organisms [START_REF] Muyssen | Mechanisms of chronic waterborne Zn toxicity in Daphnia magna[END_REF]. Concerning human health, a zinc surplus is related to oxidative stress and a contributing factor in many chronic diseases [START_REF] Walsh | Zinc: Health effects and research priorities for the 1990s[END_REF]. In urban areas, human activities mobilize large quantities of Zn into the environment. Vehiclerelated emissions (tires and brakes) and road furniture (galvanized steel) contribute to the release of zinc into the environment [START_REF] Councell | Tire-wear particles as a source of zinc to the environment[END_REF][START_REF] Legret | Evaluation of pollutant loadings in the runoff waters from a major rural highway[END_REF][START_REF] Zarcinas | Copper, lead and zinc mobility and bioavailability in a river sediment contaminated with paint stripping residue[END_REF]. Zn emissions in the road environment of about 300 metric tons per year in France [START_REF] Gouzy | Données technico-économiques sur les substances chimiques en France : Zinc et principaux composés[END_REF] are mainly due to wear and tear from tires [START_REF] Hjortenkrans | Metal emissions from brake linings and tires: Case studies of Stockholm, Sweden 1995/1998 and 2005[END_REF]. Such particles are then leached by rainwater, forming runoff with high Zn levels polluting both riverbed sediments and water [START_REF] Lee | Characteristics of a polluted artificial soil located along a motorway and effects of acidification on the leaching behavior of heavy metals (Pb, Zn, Cd)[END_REF]. Large quantities of Zn (about 150 metric tons per year in France) are also released as liquid effluents into surface water by industry and municipalities (http://prtr.ec.europa.eu/#/home).

Half of this comes from urban wastewater treatment plants (WWTP), mainly as dissolved Zn [START_REF] Gourlay-Francé | Labile, dissolved and particulate PAHs and trace metals in wastewater: Passive sampling, occurrence, partitioning in treatment plants[END_REF][START_REF] Gouzy | Données technico-économiques sur les substances chimiques en France : Zinc et principaux composés[END_REF].

Because river basins are critical components in the hydrologic cycle, the control of Zn content in surface water is a major environmental and public-health issue (EU Water Framework Directive 2000/60/EC). Accordingly, Zn is regulated and monitored by 22 member states in EU rivers [START_REF] Arle | Monitoring of Surface Waters in Germany under the Water Framework Directive-A Review of Approaches[END_REF]. In France, a good ecological status is reached in water when Zn content is below the environmental quality standard value (EQS), i.e. 7.8 µg/L (https://substances.ineris.fr/fr/page/9). For a good management of water bodies, defining Zn limits in the environment below which unacceptable effects are not expected, is insufficient. We must also consider the manifold pollution sources, the presence of variable 'naturally' occurring background input, the existence of several chemical species, some of which are more toxic than others, and the variation in abundance of such species in response to changes in physico-chemical water conditions.

With the advent of multi-collector inductively coupled plasma mass spectrometry (MC-ICP-MS), a stable-isotope variability of transition metals (Zn, Cu, etc.) has been observed in many natural samples [START_REF] Belshaw | High precision measurement of iron isotopes by plasma source mass spectrometry[END_REF][START_REF] Halliday | Recent developments in inductively coupled plasma magnetic sector multiple collector mass spectrometry[END_REF][START_REF] Maréchal | Precise analysis of copper and zinc isotopic compositions by plasma-source mass spectrometry[END_REF].

The stable-isotope compositions of Zn (δ 66/64 Zn) have been widely studied in Zn ores from various deposits leading to an average for sulphide ores of around +0.16±0.20‰ [START_REF] Sonke | Historical variations in the isotopic composition of atmospheric zinc deposition from a zinc smelter[END_REF]. The bulk silicate earth value for δ 66/64 Zn is +0.28±0.05‰ [START_REF] Chen | Zinc isotope fractionation during magmatic differentiation and the isotopic composition of the bulk Earth[END_REF] and values between +0.7‰ and +1.1‰ are generally determined for Zn-isotope compositions for carbonates [START_REF] Chen | Zn isotopes in the suspended load of the Seine River, France: Isotopic variations and source determination[END_REF][START_REF] Luck | Pb, Zn, and Cu isotopic variations and trace elements in rain[END_REF][START_REF] Pichat | Zinc isotope variations in deep-sea carbonates from the eastern equatorial Pacific over the last 175 ka[END_REF], which are higher than for other geologic materials (silicates, sulphides). The δ 66/64 Zn data for sediments, suspended particulate matter and water sampled in unpolluted rivers and lakes, i.e. the geological background for surface water (GBSW), range between +0.24‰ and +0.41‰ with a median value of +0.30‰ (n=127, [START_REF] Desaulty | Zinc isotope composition as a tool for tracing sources and fate of metal contaminants in rivers[END_REF], close to the bulk silicate earth value (+0.28±0.05‰, [START_REF] Chen | Zinc isotope fractionation during magmatic differentiation and the isotopic composition of the bulk Earth[END_REF]. Zn is not a redox-sensitive element under most natural conditions, though fractionation caused by natural processes like mineral adsorption, biotic adsorption, complexation by organic matter, and metabolic uptake by plants and microorganisms can control the isotopic composition of dissolved Zn in an aqueous environment [START_REF] Moynier | The isotope geochemistry of zinc and copper[END_REF]. The Zn-isotope fractionation introduced by industrial processing has been evaluated from anthropogenic samples. Road-traffic emissions (fuel and brake-lining and tire dust) cover a range of values depleted in heavy isotopes (-0.50‰ to +0.23‰, n=21, [START_REF] Borrok | Zinc isotopic composition of particulate matter generated during the combustion of coal and coal + tire-derived fuels[END_REF][START_REF] Dong | Isotopic signatures suggest important contributions from recycled gasoline, road dust and nonexhaust traffic sources for copper, zinc and lead in PM10 in London, United Kingdom[END_REF][START_REF] Gioia | Accurate and precise zinc isotope ratio measurements in urban aerosols[END_REF][START_REF] Souto-Oliveira | Multi-isotopic fingerprints (Pb, Zn, Cu) applied for urban aerosol source apportionment and discrimination[END_REF][START_REF] Thapalia | Zn and Cu isotopes as tracers of anthropogenic contamination in a sediment core from an Urban Lake[END_REF] compared to non-impacted rivers (GBSW +0.24‰ to +0.41‰, [START_REF] Desaulty | Zinc isotope composition as a tool for tracing sources and fate of metal contaminants in rivers[END_REF]. The δ 66/64 Zn values for liquid effluents from industry and WWTP (-0.03‰ to +0.15‰, n=12, [START_REF] Chen | Zinc isotopes in the Seine River waters, France: A probe of anthropogenic contamination[END_REF][START_REF] Desaulty | Zinc isotope composition as a tool for tracing sources and fate of metal contaminants in rivers[END_REF] are close to those from road-traffic emissions and lower than those in non-impacted rivers.

Because the isotopic composition of Zn could be used as an environmental tracer for tracking the sources of urban and industrial pollution, and its transport into water and soil, we tested using Zn isotopes for studying the human impact due to WWTP releases and road traffic emissions in a small watershed used as case study. We assessed the ability of Znisotope compositions for highlighting the major urban Zn emissions into the environment by analysing samples (water, sediment, and liquid effluent) in a small catchment with wellknown metal contributions. The Egoutier River (Loire River basin, France) rises in a pristine area and flows for about 1 km before being affected by liquid effluents from a WWTP and, farther down, by road traffic. Samples of pollution sources as well as of water and sediment along an upstream to downstream transect were taken for one year. For effluents and waters, we used both "grab" sampling and integrated sampling with passive Diffusive Gradients in Thin films (DGT) samplers. DGT use for analysing Zn-isotope compositions in water was developed and validated by a previous study [START_REF] Desaulty | Coupling DGT passive samplers and multi-collector ICP-MS: A new tool to measure Pb and Zn isotopes composition in dilute aqueous solutions[END_REF]. DGT sampling pre-concentrates metals in situ, thus avoiding a time-consuming chemical preparation and integrates potential variations of the Zn isotopic signature over time.

The study of this small watershed with its well-known metal contributions allowed assessing the extent to which Zn-isotope compositions in river water can be used for tracking anthropogenic contributions on a watershed scale. The use of a new sampling technique such as DGT for determining Zn isotope compositions to track anthropogenic contributions in surface water is also investigated.

Material and methods

Study site

The Egoutier River catchment (15 km 2 ) is located in the Loire River basin close to Orléans (France) and is part of the geological region of the Paris Basin [START_REF] Gigot | Carte géologique de la France au 1/50.000[END_REF]. [START_REF] Everbecq | Application du modèle PEGASE à l'échelle du Bassin de l'Agence[END_REF], using the PEGASE model, calculated a mean river flow of 0.025 m 3 /s (upstream) to 0.38 m 3 /s (downstream); the observed river flow during low-water periods is between 0.00014 m 3 /s and 0.00233 m 3 /s. From upstream to downstream, the bedrock consists dominantly of (1) sand and clay, (2) marl and sand, and (3) marl and limestone [START_REF] Gigot | Carte géologique de la France au 1/50.000[END_REF] (Fig. 1).

The Egoutier River flows for 4 km before joining the Loire River tributary named 'Canal of Orléans'. The study area covers a short distance of 4 km between the spring and a busy provincial road (Fig. 1). The study zone can be impacted from upstream to downstream by (1) effluent releases from a wastewater treatment plant (WWTP) in a hospital, (2) a tributary draining an oil depot and (3) traffic pollution from a busy road.

The WWTP treats the wastewater from the local departmental hospital centre (DHC), supplied in water from its own borehole. The hospital is dedicated to mental-health-care services and has no radiology equipment. It is relatively small (300 beds) and promotes ambulatory medicine, but it has an important laundry activity with a daily load of about one ton. The resulting water flow-treated by the WWTP and rejected into the Egoutier River-is not measured, but a first estimate based on water consumption gives a value of 0.002 m 3 /s. This value is of the same order of magnitude as the average release from a <10,000 PE WWTP (PE = population equivalent; https://rsde.ineris.fr/).

No metal-rich releases are referenced by the Water Agency databases in the tributary draining the nearby oil depot, whose soil is not registered as polluted by metal (http://basol.developpement-durable.gouv.fr). Traffic flow on the departmental road crossing the Egoutier at the limit of the study area, is large (50,000 vehicles/day) and similar to that of an average highway [START_REF] Maurin | Carte] Circulation : quels sont les axes les plus empruntés dans l'agglomération d'Orléans ? La république du Centre[END_REF].

Sampling, preparation and analytical techniques

Sample types from the Egoutier River catchment

Both liquid and solid samples from the Egoutier River were analysed for this study (Tables 1 and2). Samples taken in 2014 and 2015 included dissolved fraction from upstream to downstream (# spring, 0, 1, 3, 4, 4bis, 6, 7, 8 and final) and its tributaries (# 2-6, 2 and 5) (Fig. 1). Samples # 2-6 and # 2 were taken in Egoutier tributary upstream and downstream from the DHC WWTP releases. Sampling point # 5 is located in the Egoutier tributary draining the oil depot. Sampling point # final is located at the downstream end of the study area in the Egoutier River, where runoff draining the departmental road is discharged (Fig. 1). During low-flow periods , the water level was too low for sampling at points # spring, 0 and 1. In addition to surface waters, groundwater samples without anthropogenic contribution were also collected from the DHC borehole (# drilling) "upstream" of the treatment plant. Raw wastewater and plant-treated wastewater were collected directly in the DHC's WWTP.

Passive DGT samplers were also deployed for two weeks (DHC WWTP, # 2-6) and three weeks (# 2,3,5,4bis,Final) in the DHC WWTP and the Egoutier River. In a DGT, the labile metal fraction is fixed in a chelex resin layer after diffusion through a polyacrylamide gel [START_REF] Davison | In-situ speciation measurements of trace components in natural waters using thin-film gels[END_REF]. Low-flow rates of the river can introduce a significant error in the quantification of metal concentrations due to the presence of a diffusion boundary layer (DBL), which increases the thickness of the diffusion layer [START_REF] Garmo | Estimation of diffusive boundary layer thickness in studies involving diffusive gradients in thin films (DGT)[END_REF][START_REF] Warnken | Accuracy of the diffusive gradients in thin-films technique: Diffusive boundary layer and effective sampling area considerations[END_REF]. Consequently, DGT were used only for determining the Zn isotope compositions, which are not impacted by the presence of DBL [START_REF] Desaulty | Coupling DGT passive samplers and multi-collector ICP-MS: A new tool to measure Pb and Zn isotopes composition in dilute aqueous solutions[END_REF].

Sediment samples were collected in April 2015 from the main stream (# spring, 1, 3, 4bis and final) and in a tributary (# 2) (Fig. 1). In addition, road particles located close to the Egoutier in sampling point # final were also collected.

Sampling methodology

During each sampling, temperature, pH, redox potential, dissolved oxygen and conductivity of the water were measured in the middle of the river using multi-parameter probes (MultiLine Multi 3620 IDS).

All liquid samples, collected with an acid-cleaned plastic bottle, were filtered immediately in the field using a 0.45 µm cellulose acetate filter. The fraction passing the filter is defined as the dissolved fraction. Solutions for elemental and isotopic-ratio analyses were acidified to pH ~2 with sub-boiled concentrated HNO3. After a rinsing step in "Milli-Q" water, passive DGT samplers were fixed on polyethylene holder plates designed by BRGM. The holder plates were ballasted with weights to prevent the DGT from floating and placed in a polyethylene cage to avoid damage. The whole assembly was tied with a rope to tree branches above the river and immersed in the water.

Sediments were sampled with a shovel in the middle of the river to a depth of around 10 centimetres. The samples were directly sieved at 2 mm with the sampling water and stored in plastic bags. Road-particle samples were collected with a plastic spatula, sieved at 2 mm and stored in plastic bottles. They were taken during days without rainfall 24 h before sample collection. Daily rainfall from 1 October to 30 November 2015 was recorded by the Meteo France weather station in Orléans (https://donneespubliques.meteofrance.fr).

Reagents and materials

All plastic and Teflon equipment for this study was acid-cleaned before use. All acids were purified by sub-boiling distillation before use. The water was distilled "Milli-Q" water with a resistivity of 18.2 MΩ.cm (Millipore®). Anion exchange resin AGMP-1 (100-200 mesh, chloride form, BioRad®) was used for Zn purification. The passive DGT sampling devices were purchased from DGT®Research. Detailed descriptions of the sampler can be found at DGT Research's homepage (http:// www.dgtresearch.com/).

Preparation of liquid samples and DGT samples

The liquid samples were diluted in 0.5 M HNO3 for elemental analysis. Only samples with a Zn content over 20 µg/L were prepared for isotopic analysis. A volume corresponding to about 1 µg of Zn was evaporated to dryness on a hot plate in the cleanroom. This step was repeated twice successively with 100 µL concentrated HNO3 and HCl. The DGT units were disassembled and the resin gels were transferred into polypropylene tubes in the cleanroom.

We used 1N HNO3 to extract metals from the chelex resin using the protocol described by [START_REF] Desaulty | Coupling DGT passive samplers and multi-collector ICP-MS: A new tool to measure Pb and Zn isotopes composition in dilute aqueous solutions[END_REF]. The solution was evaporated to dryness on a hot plate and reserved for isotopic analysis. For DGT, the procedural "blank" was <10 ng.

Solid sample preparation

The solid samples (sediments and road particles) were dried for 3 days at 38 °C in an oven.

Sediments and road particles were separated into two aliquots for determining "bulk" compositions (total fraction) and for the extraction experiment (mobile fraction). For "bulk" compositions, the samples were crushed in an agate ball mill to a particle size of about 100 µm. Part of the sample was reserved for mineralogical analysis. Several grams of crushed sample were calcined at 550 °C and about 500 mg was dissolved in concentrated acids (HNO3, HClO4, HF, HCl) on a hot plate in the cleanroom [START_REF] Petit | Development of Cu and Zn isotope MC-ICP-MS measurements: Application to suspended particulate matter and sediments from the scheldt estuary[END_REF]. After evaporation to dryness, the residue was diluted in 0.5 M HNO3 and prepared for elemental and isotopic analysis as liquid samples. For the extraction experiment, 4 g of sediment or road particles were leached with 80 mL of 0.2 M HCl and shaken for 2:30 hrs. The pH value was checked before and after shaking to verify the extraction efficiency. After extraction, the solid residue was separated by centrifugation and oven-dried at 38 °C. The supernatant was filtered with a 0.45 µm nylon filter. The quantity of acid-extractable matter (AEM, expressed in percent, [START_REF] Négrel | The labile fraction of suspended matter in the Loire River (France): multi-element chemistry and isotopic (Rb-Sr and C-O) systematics[END_REF], representing the mobile fraction, was determined for each sample by the difference in weight between the initial dried sample (mini, expressed in grams) and the dried residue after acid extraction (mresidue, expressed in grams), and shown as a percentage of the total matter content (mini, expressed in grams) (see Eq. ( 1)).

(1) Uncertainties on the AEM determination are estimated to be close to a few percent [START_REF] Négrel | The labile fraction of suspended matter in the Loire River (France): multi-element chemistry and isotopic (Rb-Sr and C-O) systematics[END_REF]. The supernatant was prepared for elemental and isotopic analysis as liquid samples in the cleanroom. The residue was reserved for mineralogical analysis.

Analytical techniques

Major elements were analysed with a Horiba Scientific Ultima ICP-OES (inductively coupled plasma optical emission spectrometer). The precision for major element concentrations was generally better than 10%. The trace-element concentrations were measured with a Thermo Fisher Scientific X Series 2 ICP-MS (ICP mass spectrometer). Analytical quality was controlled by internal-standard (In and Re) and regular international-geo-standard (SLRS4) measurements. The precision for trace-element concentrations was generally better than 5%.

The mineral-phase compositions of sediments and their residues after acid leaching were determined by XRD (X-ray diffraction). Powdered samples were analysed with a Siemens D5000 diffractometer equipped with a cobalt source (Cokα radiation, λ=1.789 Å).

Diffraction patterns were recorded over an angular range from 4 to 84°2thêta with a step size of 0.02°2thêta. Quantitative analyses were done with the Rietveld refinement method [START_REF] Rietveld | A profile refinement method for nuclear and magnetic structures[END_REF][START_REF] Rietveld | Line profiles of neutron powder-diffraction peaks for structure refinement[END_REF]. The precision of the results, generally between 3 and 5%, was estimated from the refinement quality (accuracy of fit) obtained during modelling of the diffractogram and given by the software.

Before isotopic analyses, anion-exchange purification was necessary for separating Zn from co-existing matrix elements (Na, K, Ca and traces of Ba, Cu, Ni and Ti) with a protocol adapted from [START_REF] Borrok | Separation of copper, iron, and zinc from complex aqueous solutions for isotopic measurement[END_REF]. After evaporation to dryness, the sample was put in a mixture of 10 M HCl and 0.001% H2O2 and passed through an anion-exchange column loaded with macroporous AG MP-1 resin. The unwanted matrix elements were eluted successively in HCl, Zn was then eluted in diluted HNO3, evaporated to dryness and dissolved in 0.5 M HNO3 for isotopic measurements. For DGT, one elution was enough to purify Zn, while for other samples the elution step was repeated for complete elimination of the rest of the matrix. For such chemical purification, the procedural "blank" was <5 ng. Zn recovery from this protocol was checked by analysing one aliquot before and after the chemical separation by ICP-MS and recoveries were consistently close to 100%. Total procedural blanks (from field to mass spectrometry analysis) were measured to verify the cleaning procedure. Procedural blanks are generally less than 20 ng, representing less than 2% of the zinc mass analysed.

Zn-isotope compositions were measured at a concentration of 300 µg/L in a Thermo Fisher Scientific Neptune MC-ICP-MS (Multi-collector ICP-MS). The Zn data were normalized to Cu using an exponential mass-fractionation law; instrument drift was corrected by standard bracketing with a JMC 3-0749 L solution called "JMC Lyon" [START_REF] Maréchal | Precise analysis of copper and zinc isotopic compositions by plasma-source mass spectrometry[END_REF] (see Eq. ( 2)). The Zn-isotope composition of each sample was expressed in δ-notation relative to the mean value of the bracketing Zn standard ("JMC Lyon"):

(2)

The quality of Zn-isotope analyses was controlled by regular measurements of "inhouse" standards, whose long-term reproducibility was 0.04‰ (2σ) for δ 66/64 Zn. The external reproducibility (2σ) reported in the various figures and tables was typically ±0.03‰, calculated by measuring the same sample multiple times over the many analytical sessions.

Results

Data are listed in Tables 1 and2. We first examine the Zn contents and isotopic compositions for wastewater and effluent from the DHC WWTP, and then we describe the data from the Egoutier samples (water, sediments, and road particles) collected along an upstream/downstream profile.

Zn contents and isotopic compositions for raw wastewater and wastewater treated by the DHC WWTP

The Zn contents and δ 66/64 Zn values for raw wastewater, wastewater treated by DHC's WWTP and literature data [START_REF] Chen | Zinc isotopes in the Seine River waters, France: A probe of anthropogenic contamination[END_REF][START_REF] Desaulty | Zinc isotope composition as a tool for tracing sources and fate of metal contaminants in rivers[END_REF]) are shown on Figure 2. The δ 66/64 Zn values for DHC's WWTP effluents range from +0.03‰ to +0.17‰ and are close to data from previous studies (-0.03‰ to +0.15‰).

Dissolved Zn contents are generally twice as high in treated effluents as in raw wastewater (Fig. 2). Treated wastewater from industrial WWTPs and the DHC one has lower δ 66/64 Zn values than raw wastewater. Moreover, the Zn-isotope composition of the DHC WWTP effluents obtained with a passive DGT sampler deployed for 14 days is lower (+0.03‰) than the value for treated wastewaters obtained by "grab" sampling (+0.08‰ and +0.17‰), and close to the values of -0.03‰ and +0.08‰ obtained by [START_REF] Chen | Zinc isotopes in the Seine River waters, France: A probe of anthropogenic contamination[END_REF].

The δ 66/64 Zn data for WWTP effluents (n=15) are shown as a boxplot giving interquartile values between +0.07‰ and +0.14‰ and a median value of +0.10‰ (Fig. 2).

The δ 66/64 Zn values for effluents analysed both by us and in previous studies are below those obtained for non-impacted water and sediments (GBSW, +0.24‰ to +0.41‰, Desaulty and Petelet-Giraud, 2020; Fig. 2).

The Egoutier River samples

The Zn contents and isotopic compositions for samples collected along the Egoutier transect are shown on Figures 3 and4.

Zn contents and isotopic compositions for water

The dissolved Zn contents are quite variable between sampling campaigns, though it is possible to identify main trends (Fig. 3). For all samples collected downstream from the DHC WWTP, the dissolved Zn contents in water are higher than the EQS value (7.8 µg/L) (Table 1). Zn concentrations for sampling point # 3 downstream of the WWTP releases are generally ten times higher than those in the spring. The tributary draining the oil depot (# 5) has a Zn content of around 3 µg/L, close to that of the spring (~4 µg/L), and no increase in Zn content is observed downstream (# 6). For the sampling point just after the road-traffic contribution (# final), there is no clear rise in Zn contents compared to points # 6, 7 and 8 farther upstream.

The Zn-isotope compositions for water in "grab" samples (07-Oct-15, 21-Oct-15) and using the passive DGT sampler are shown on Figure 4. For the same point, the δ 66/64 Zn values are rather variable from one sampling day to the next and commonly slightly higher than the value obtained by DGT. Despite these differences, main trends are seen in the δ 66/64 Zn values along the upstream/downstream profile. Before the DHC WWTP, the Znisotope compositions (+0.22‰ to +0.26‰) are close to the GBSW median value (+0.30‰, Desaulty and Petelet-Giraud, 2020), while the downstream points have lighter isotopic signatures (~ +0.15‰, Fig. 4). The sampling point in the Egoutier tributary draining the oil depot (# 5) is an exception with a δ 66/64 Zn value (+0.27‰) close to the GBSW median value (+0.30‰, Desaulty and Petelet-Giraud, 2020).

Zn mineral compositions of the total and mobile fractions in sediments

The composition of major elements and mineral phases distinguishes three sediment facies that mirror the underlying geology (Table S-1; Supplemental online material).

Sediments sampled in the first "# spring" sampling point located in geological layer "Sand and Clay of Sologne" are composed of silicates and clay minerals (50% each). The sediments # 1, 2, 3 and 4bis collected in "Marl and Sand of the Orléanais" are mainly composed of silicates (>75% by weight) with a small proportion of clay minerals (<30% by weight). The sediments sampled at # final located in "Marl and Limestone of the Orléanais" have a mineral composition close to the other sediments (# 1, 2, 3 and 4bis) with the addition of calcite (12% by weight). The road particles sampled at sampling point # final have a mineralogical composition close to the sediment collected in the same place.

The mineral compositions of residues after acid leaching in 0.2N HCl are shown in Table S-1. The silicate and clay minerals occur in the same proportions in the sediments and their residues after acid leaching (Table S-1). Calcite, present in a large proportion in samples # final (12%) and # road particles (16%) is missing in their residues (Table S-1).

Zn contents and isotopic compositions of the total and mobile fractions in sediments

Zn-enriched (>90 mg/kg) sediments were sampled at points # spring, 2, 3 and final (Table 2). The road particles collected in # final have a higher Zn content (919 mg/kg) than the sediments (<300 mg/kg; Fig. 3). The δ 66/64 Zn values for sediments along the upstream-todownstream profile are shown in Figure 4. As for the water samples, the sediments located upstream from DHC WWTP have Zn-isotope compositions (+0.27‰ and +0.28‰) close to the GBSW median value (+0.30‰, Desaulty and Petelet-Giraud, 2020), while the downstream points have lighter isotopic signatures (~ +0.15‰). The δ 66/64 Zn value for road particles (+0.16‰) is also depleted in heavy isotopes relative to the +0.30‰ GBSW median value and close to samples reflecting the road-traffic pollution presented as a boxplot with interquartile values between +0.08‰ and +0.20‰ (n=21, [START_REF] Borrok | Zinc isotopic composition of particulate matter generated during the combustion of coal and coal + tire-derived fuels[END_REF][START_REF] Dong | Isotopic signatures suggest important contributions from recycled gasoline, road dust and nonexhaust traffic sources for copper, zinc and lead in PM10 in London, United Kingdom[END_REF][START_REF] Gioia | Accurate and precise zinc isotope ratio measurements in urban aerosols[END_REF][START_REF] Souto-Oliveira | Multi-isotopic fingerprints (Pb, Zn, Cu) applied for urban aerosol source apportionment and discrimination[END_REF][START_REF] Thapalia | Zn and Cu isotopes as tracers of anthropogenic contamination in a sediment core from an Urban Lake[END_REF]; Fig. 2).

The Zn contents and isotopic compositions for the mobile sediment and road-particle fractions obtained by 0.2N HCl leaching are shown on Figures 3,4 and 5. Fig. 5 shows for major elements (Al, K, SiO2, Fe, Ca) the proportion of elements released by the leaching of sediments and road particles ('mobile fraction') compared to their "bulk" compositions ('total fraction'), as well as the AEM (%) values. The AEM proportion ranges from 2% to 16% and displays a good relationship with calcite abundance (Table S-1). Few quantities of Al, SiO2 and K are taken into solution after the extraction in 0.2 N HCl (0.1-4.5%), and only a small proportion of Fe (3-20%) is dissolved by the extraction (Fig. 5). All Ca in samples # final and # road particles is released in the mobile fraction. Less than 20% of Zn is liberated by acid extraction for the non-polluted sampling points (# spring, 1), while a larger amount (>60%) is released for the points just after the WWTP and road contributions (# 2 and final). The mobile sediment fraction, with its lower Zn concentration, was collected in sampling points # spring and # 1 (<15 mg/kg) (Table 2). The Zn content in the mobile fraction for sediments sampled just after the DHC WWTP in the Egoutier tributary (# 2) and in the main stream (# 3) is 3 to 11 times higher than in sample # 1. The sediment leachate collected just after the road traffic pollution (# final) contains six times more Zn than the upstream sediment # 4bis. The highest Zn content was found in the mobile fraction of road particles (751 mg/kg). As for the water and "bulk" sediments, the Zn-isotope composition of the mobile sediment fraction upstream of the DHC WWTP is higher (+0.40-+0.53‰) than in downstream points (~+0.18‰) (Fig. 4).

The Zn-isotope compositions for the total and mobile sediment fractions are presented in Table 2; except for sampling points # spring and # 1, the δ 66/64 Zn values being similar to those of the total and mobile fractions.

Discussion

Figure 2 shows, from literature data and our DHC WWTP samples, that the δ 66/64 Zn values for WWTP effluents (-0.03‰ to +0.17‰) are depleted in heavy isotopes compared to the raw wastewater (+0.13‰ to +0.28‰). Whereas wastewater undergoes various treatment stages (primary settling, secondary activated sludge and tertiary flocculation by FeCl3) to decrease particulate metal concentrations in the final effluent, the dissolved-metal content tends to increase along these treatment steps [START_REF] Buzier | Trace metal speciation and fluxes within a major French wastewater treatment plant: Impact of the successive treatments stages[END_REF][START_REF] Gourlay-Francé | Labile, dissolved and particulate PAHs and trace metals in wastewater: Passive sampling, occurrence, partitioning in treatment plants[END_REF]. The dissolution of iron salts added during dephosphorization treatment, e.g. FeCl3 containing high trace-metal contents, can explain this increasing dissolved metal concentration during the treatment steps [START_REF] Gourlay-Francé | Labile, dissolved and particulate PAHs and trace metals in wastewater: Passive sampling, occurrence, partitioning in treatment plants[END_REF]. Therefore, the difference in δ 66/64 Zn values between raw wastewater and WWTP effluents is likely due to the dissolution of chemical components rich in Zn and depleted in heavy isotopes added during treatment [START_REF] Desaulty | Zinc isotope composition as a tool for tracing sources and fate of metal contaminants in rivers[END_REF]. With only small differences of δ 66/64 Zn in WWTP effluents (+0.07‰ and +0.14‰, interquartile values, n=15, [START_REF] Chen | Zinc isotopes in the Seine River waters, France: A probe of anthropogenic contamination[END_REF][START_REF] Desaulty | Zinc isotope composition as a tool for tracing sources and fate of metal contaminants in rivers[END_REF] this study) and road and traffic pollution (+0.08‰ to +0.20‰, interquartile values, n=21, [START_REF] Borrok | Zinc isotopic composition of particulate matter generated during the combustion of coal and coal + tire-derived fuels[END_REF][START_REF] Dong | Isotopic signatures suggest important contributions from recycled gasoline, road dust and nonexhaust traffic sources for copper, zinc and lead in PM10 in London, United Kingdom[END_REF][START_REF] Gioia | Accurate and precise zinc isotope ratio measurements in urban aerosols[END_REF][START_REF] Souto-Oliveira | Multi-isotopic fingerprints (Pb, Zn, Cu) applied for urban aerosol source apportionment and discrimination[END_REF][START_REF] Thapalia | Zn and Cu isotopes as tracers of anthropogenic contamination in a sediment core from an Urban Lake[END_REF], it is difficult to discriminate road traffic from effluent pollution in the environment by using Zn-isotope signatures (Fig. 2). However, with the δ 66/64 Zn depleted in heavy isotopes for these anthropogenic contributions compared to a non-impacted signature (GBSW, +0.24‰ to +0.41‰), it is possible to track anthropogenic Zn in the environment.

Hereafter, we assess the extent to which Zn-isotope compositions can be used for tracking both source and transport of anthropogenic Zn in a watershed.

Evidence of mixing in water

For liquid samples, Figure 6, showing δ 66/64 Zn values versus 1/Zn ratios, defines the mixture of two components with different isotopic compositions and Zn contents as a straight-line mixing curve [START_REF] Faure | Principles of Isotope Geology, Second Edition[END_REF]. While this figure should be interpreted with caution, it clearly shows a mixing line between a 'geogenic' component composed of groundwater free of anthropogenic contribution (# drilling) and an 'anthropogenic' one (WWTP effluents of 07-Oct-15). Almost all value data points fall between these two endmembers. The water in sampling points # 2-7 and # 3 is composed of almost 40% of liquid effluent, and the "grab" samples collected on 07-Oct-15 (# 2) and 21-Oct-15 (# 7, 8) contain between 4% and 10% anthropogenic input. This result shows that WWTP-effluent input is still seen three kilometres downstream in water. The # final sample collected just downstream of the road contains between 4% and 10% anthropogenic input, consistent with a road-traffic contribution (Fig. 6).

Samples # 2 and 4-bis collected on 21-Oct-15 and the WWTP effluents sampled the same day, plot outside this mixing line, which might be explained by a contribution from another Zn source, though this seems unlikely as the metal sources are well-known for this watershed. Although the differences in pH, redox conditions and conductivity of the water compared to other samples are not clear, local physico-chemical processes due to biological uptake or abiotic mechanisms controlling the distribution of metals between dissolved and particulate phases (adsorption, dissolution, precipitation, etc.) may explain the deviation of samples # 2 and 4-bis from the mixing line on Figure 6.

Evidence of mixing in sediments: mobile and total fractions

For the total fraction in sediments, the variation in Zn content between the various sediments sampled in the upstream/downstream profile can be due to a disparity in mineral compositions, rather than to anthropogenic pollution. The mineral phases that compose the sediment have naturally differing metal contents. Studies based on sequential-extraction experiments have shown that aluminium, iron oxides, clay minerals and carbonates play an important role in controlling the Zn content in sediments [START_REF] Accomasso | A study on the reproducibility of Tessier's extractions in a fluvial sediment and a comparison between different dissolution procedures in a reference material[END_REF][START_REF] Belzunce-Segarra | Chemical forms of heavy metals in surface sediments of the San Simón inlet, Ría de Vigo, Galicia[END_REF][START_REF] Bordas | A critical evaluation of sample pretreatment for storage of contaminated sediments to be investigated for the potential mobility of their heavy metal load[END_REF][START_REF] Dawson | Speciation of heavy metals in floodplain and flood sediments: a reconnaissance survey of the Aire Valley, West Yorkshire, Great Britain[END_REF][START_REF] Kwon | Sedimentation pattern and sediments bioavailability in a wastewater discharging area by sequential metal analysis[END_REF][START_REF] Li | Heavy metals in coastal wetland sediments of the Pearl River Estuary, China[END_REF][START_REF] Tessier | Sequential extraction procedure for the speciation of particulate trace metals[END_REF]Usero et al., 1998). To avoid the effect of disparity in mineralogical composition and to highlight only the upstream/downstream variations, the Zn contents were normalized to those elements in the mineral phases where Zn is naturally the more concentrated or associated, i.e. aluminium, iron and calcium. These normalized Zn-content ratios (Zn/ (Al+Fe+Ca)*10 4 ) are shown on Figure 3 along an upstream/downstream profile. This highlights the Zn enrichment for sediment in sampling point # 3 compared to the upstream ones (# spring, 1; Fig. 3). The graph of δ 66/64 Zn values versus (Al+Fe+Ca)/Zn*10 -4 ratios shows that most samples are a mix between two endmembers: (1) a natural input of sediments sampled upstream of any anthropogenic contributions (sediment # 1) with a mineralogical composition close to that of the downstream samples; and (2) an anthropogenic input with a low (Al+Fe+Ca)/Zn*10 -4 ratio (i.e. a high Zn content) and low δ 66/64 Zn value (Fig. 7). Without analyses of the particulate matter in WWTP effluents, we cannot precisely estimate the parts of the endmembers in sediments, but the anthropogenic input is consistent with the δ 66/64 Zn value of WWTP effluents and especially with the DGT data (+0.03‰).

The sediment most impacted by anthropogenic pollution is sample # 2 (Fig. 7). The # final sediment seems to contain several contributions: geogenic, road traffic and WWTP effluents. Like the water in the same sample (Fig. 6), sediment # 4bis plots outside the mixing line (Fig. 7); this cannot be explained by different mineralogical compositions (Table S1), but may be due-as for water-to local physico-chemical processes (abiotic or biotic mechanisms).

By comparing the elemental and mineralogical compositions of products with residues from the extraction experiment, we can determine the mineral phases controlling the mobile sediment fraction (Fig. 5; Table S1). The results show that calcite is completely leached, whereas silicate and clay minerals are not extracted by the protocol. The small proportion of Fe that is dissolved by extraction probably comes from hydrous Fe oxides. As shown previously by [START_REF] Négrel | The labile fraction of suspended matter in the Loire River (France): multi-element chemistry and isotopic (Rb-Sr and C-O) systematics[END_REF] and [START_REF] Négrel | Investigating the sources of the labile fraction in sediments from silicatedrained rocks using trace elements, and strontium and lead isotopes[END_REF], using a diluted acid reagent releases non-residual trace elements. In ascending order of mobility, they are those associated with hydrous Fe oxides (reducible fraction), those bound to carbonates (acidsoluble fraction), and those adsorbed on various minerals (exchangeable fraction) that are liberated in the mobile fraction. The upstream/downstream profile of Zn contents for the mobile sediment fraction, highlights the contamination after the DHC WWTP and the departmental road (Fig. 3). For sediments located downstream of the WWTP (# spring, 1) only a small fraction of the total Zn (<20%) is dissolved by extraction in 0.2N HCl (Fig. 5), which explains the different δ 66/64 Zn values for total and mobile fractions of these samples.

Figure 8, showing the δ 66/64 Zn values versus 1/Zn ratios for the mobile fraction, highlights the anthropogenic input for sediment located downstream of the WWTP effluents.

The high δ 66/64 Zn values for sample # final relative to other sediments is likely due to the fact that this sample contains a large proportion of calcite with high δ 66/64 Zn values (+0.7‰ to +1.1‰, [START_REF] Chen | Zn isotopes in the suspended load of the Seine River, France: Isotopic variations and source determination[END_REF][START_REF] Luck | Pb, Zn, and Cu isotopic variations and trace elements in rain[END_REF][START_REF] Pichat | Zinc isotope variations in deep-sea carbonates from the eastern equatorial Pacific over the last 175 ka[END_REF], which tends to hide the anthropogenic contribution to the isotopic signature.

Using δ 66/64 Zn values for tracking Zn origin and fate at the watershed scale

Using Zn-isotope signatures, the WWTP-effluent input is clear in water and sediments sampled downstream of the DHC WWTP (Fig. 678). This allows tracing the fate of this contribution at the scale of the watershed. At sampling point # 2 in the Egoutier tributary, just downstream from the DHC WWTP outlet, the sediment is the most impacted whereas the water contains less than 10% anthropogenic contribution (Fig. 678). Of the other liquid samples, # 2-7 and # 3 contain 40% anthropogenic contribution, but sediment from sampling point # 3 is less affected. The higher contamination in water than in sediment with increasing distance from the pollution source is due to the fact that dissolved Zn is more easily transported in water than particulate Zn. Because of the low clay mineral content (Table S1), dissolved Zn is weakly fixed by adsorption on sediment, which explains that water 2.5 km after the DHC (# 8) still contains 4% anthropogenic input (Fig. 6). Farther downstream, the δ 66/64 Zn value obtained by DGT for sample # 5 (+0.27‰) in water draining the oil depot, is close to the geogenic signature (Fig. 4), confirming the fact that no metal-rich effluents are released by the oil depot.

For the most downstream sample affected by road traffic (# final), due to the similarity of δ 66/64 Zn values in WWTP effluents and in road traffic contribution, highlighting of the road traffic contribution is difficult. Here, the use of DGT sampling has proven to be a major asset.

The water sampled on 21-Oct-15 was collected during a clear day, without precipitation several days before sample collection. A major rainfall event (21.6 mm) occurred during the DGT deployment period. During this event, the particles from road traffic were leached by rainwater, forming runoff with high Zn levels and low δ 66/64 Zn that flowed into the Egoutier. This rainfall event was reflected in the DGT data, the Zn-isotope composition having a stronger anthropogenic contribution (+0.13‰) than the "grab" samples (+0.19‰; Fig. 4).

Conclusions and perspectives

Our study investigated the extent to which Zn-isotope composition can be used for tracking anthropogenic pollution (here WWTP effluent releases and road traffic contribution), at the scale of a small watershed. The use of δ 66/64 Zn values in water and sediments along an upstream/downstream profile shows the anthropogenic input of WWTP effluents, by comparing their values to those of unpolluted water and sediment. A main feature of the δ 66/64 Zn values is the WWTP-effluent contribution, which is still seen several kilometres downstream in water. Dissolved anthropogenic pollution is easily transported along the transect and only slightly fixed by adsorption on sediments, probably due to their low claymineral contents. In addition, because of the fluctuating levels of metal contaminants in anthropogenic releases, especially due to the leaching of road particles, the use of passive DGT samplers that integrate the δ 66/64 Zn values over time is a suitable method for highlighting a discontinuous anthropogenic contribution in water.

After defining the Zn-isotope systematics of natural and anthropogenic endmembers at the scale of this small watershed, the next step will be to expand the tracing of such inputs by combining the input from other trace elements. Anthropogenic releases into the environment commonly mix a combination of various elements (Cu, Pb, etc.). The monitoring of trace elements will help identifying the various anthropogenic contributions in the different compartments of a river. Moreover, introducing other isotopic systematics will help studying the origin and fate of contaminants at the watershed scale. For example, Pb isotopes are direct tracers of the origin of lead in the dissolved and solid phases, whereas Cu isotopes are strongly influenced by fractionation processes. Using a combination of carefully selected metal isotopes in concert with trace-element compositions would take us further in understanding the fate of metals in the different compartments of a river basin, by tracking various sources and constraining physical and chemical processes governing the distribution of solid and dissolved phases. [START_REF] Gigot | Carte géologique de la France au 1/50.000[END_REF]. The prospective sources of Zn contamination, found in the study area, are: (1) Effluents from a wastewater treatment plant in a hospital centre (DHC WWTP),

(2) Tributary draining an oil depot, and (3) Pollution due to heavy departmental road traffic. Zn concentration for DGT is estimated from analyses of "grab" samples. Values from previous studies for wastewater and effluents from industries, and large-and medium-sized WWTPs are also presented [START_REF] Chen | Zinc isotopes in the Seine River waters, France: A probe of anthropogenic contamination[END_REF][START_REF] Desaulty | Zinc isotope composition as a tool for tracing sources and fate of metal contaminants in rivers[END_REF].The δ 66/64 Zn values for all WWTP effluents (n=15) are shown as a purple boxplot [START_REF] Chen | Zinc isotopes in the Seine River waters, France: A probe of anthropogenic contamination[END_REF][START_REF] Desaulty | Zinc isotope composition as a tool for tracing sources and fate of metal contaminants in rivers[END_REF]this study). The δ 66/64 Zn values for samples reflected the road-traffic pollution (n=21) are shown as a grey boxplot [START_REF] Borrok | Zinc isotopic composition of particulate matter generated during the combustion of coal and coal + tire-derived fuels[END_REF][START_REF] Dong | Isotopic signatures suggest important contributions from recycled gasoline, road dust and nonexhaust traffic sources for copper, zinc and lead in PM10 in London, United Kingdom[END_REF][START_REF] Gioia | Accurate and precise zinc isotope ratio measurements in urban aerosols[END_REF][START_REF] Souto-Oliveira | Multi-isotopic fingerprints (Pb, Zn, Cu) applied for urban aerosol source apportionment and discrimination[END_REF][START_REF] Thapalia | Zn and Cu isotopes as tracers of anthropogenic contamination in a sediment core from an Urban Lake[END_REF]. The geological background for surface water "GBSW", ranging between +0.24‰ and +0.41‰ with a median value of +0.30‰, is shown as a blue line [START_REF] Desaulty | Zinc isotope composition as a tool for tracing sources and fate of metal contaminants in rivers[END_REF]. -15). Different percentages for the anthropogenic contribution are 4%, 10%, 20%, 40% and 80%. The blue "GBSW" line is the geological background for surface water, ranging between +0.24‰ and +0.41‰ with a median value of +0.30‰ [START_REF] Desaulty | Zinc isotope composition as a tool for tracing sources and fate of metal contaminants in rivers[END_REF]. The grey line covers samples describing road traffic emissions (fuel, brake lining, tire) [START_REF] Borrok | Zinc isotopic composition of particulate matter generated during the combustion of coal and coal + tire-derived fuels[END_REF][START_REF] Dong | Isotopic signatures suggest important contributions from recycled gasoline, road dust and nonexhaust traffic sources for copper, zinc and lead in PM10 in London, United Kingdom[END_REF][START_REF] Gioia | Accurate and precise zinc isotope ratio measurements in urban aerosols[END_REF][START_REF] Souto-Oliveira | Multi-isotopic fingerprints (Pb, Zn, Cu) applied for urban aerosol source apportionment and discrimination[END_REF][START_REF] Thapalia | Zn and Cu isotopes as tracers of anthropogenic contamination in a sediment core from an Urban Lake[END_REF]. The blue "GBSW" line is the geological background for surface water that ranges between +0.24‰ and +0.41‰ with a median value of +0.30‰ [START_REF] Desaulty | Zinc isotope composition as a tool for tracing sources and fate of metal contaminants in rivers[END_REF]. The grey and purple lines represent, respectively, the samples describing road traffic emissions (fuel, brake linings, tires) and WWTP effluents [START_REF] Borrok | Zinc isotopic composition of particulate matter generated during the combustion of coal and coal + tire-derived fuels[END_REF][START_REF] Chen | Zinc isotopes in the Seine River waters, France: A probe of anthropogenic contamination[END_REF][START_REF] Desaulty | Zinc isotope composition as a tool for tracing sources and fate of metal contaminants in rivers[END_REF][START_REF] Dong | Isotopic signatures suggest important contributions from recycled gasoline, road dust and nonexhaust traffic sources for copper, zinc and lead in PM10 in London, United Kingdom[END_REF][START_REF] Gioia | Accurate and precise zinc isotope ratio measurements in urban aerosols[END_REF][START_REF] Souto-Oliveira | Multi-isotopic fingerprints (Pb, Zn, Cu) applied for urban aerosol source apportionment and discrimination[END_REF][START_REF] Thapalia | Zn and Cu isotopes as tracers of anthropogenic contamination in a sediment core from an Urban Lake[END_REF]this study). Fig. 8. δ 66/64 Zn versus 1/Zn ratio for the mobile fraction of sediments and road particles from the Egoutier watershed. The blue "GBSW" line is the geological background for surface water, ranging between +0.24‰ and +0.41‰ with a median value of +0.30‰. The grey and purple lines represent, respectively, the samples describing the road traffic emissions (fuel, brake linings, tires) and WWTP effluents [START_REF] Borrok | Zinc isotopic composition of particulate matter generated during the combustion of coal and coal + tire-derived fuels[END_REF][START_REF] Chen | Zinc isotopes in the Seine River waters, France: A probe of anthropogenic contamination[END_REF][START_REF] Desaulty | Zinc isotope composition as a tool for tracing sources and fate of metal contaminants in rivers[END_REF][START_REF] Dong | Isotopic signatures suggest important contributions from recycled gasoline, road dust and nonexhaust traffic sources for copper, zinc and lead in PM10 in London, United Kingdom[END_REF][START_REF] Gioia | Accurate and precise zinc isotope ratio measurements in urban aerosols[END_REF][START_REF] Souto-Oliveira | Multi-isotopic fingerprints (Pb, Zn, Cu) applied for urban aerosol source apportionment and discrimination[END_REF][START_REF] Thapalia | Zn and Cu isotopes as tracers of anthropogenic contamination in a sediment core from an Urban Lake[END_REF]this study).

FiguresFig. 1 .

 1 Figures

Fig. 2 .

 2 Fig. 2. Zn content versus δ 66/64 Zn for samples from the DHC WWTP (raw wastewater and effluents).

Fig. 3 .

 3 Fig. 3. Zn contents for samples from the Egoutier watershed collected along an upstream/downstream profile: (a) Dissolved Zn concentrations for river water and WWTP effluents, (b) Zn contents and normalized Zn-content ratios (Zn/(Al+Fe+Ca)*10 4 ) for total fraction of sediments and road particles, (c) Zn contents for mobile fraction from sediments and road particles. The location of proven and possible sources of Zn pollution is shown on the upstream/downstream profile (DHC WWTP, tributary oil depot and road traffic). For river water and WWTP effluents, the sampling campaigns during highflow periods (08-Dec-15, 11-Dec-14, 18-Feb-15, 09-Apr-15) and low-flow periods (07-Oct-15, 21-Oct-15, 02-Nov-15) are shown by symbols in various colours. The red bar represents the values of Zn concentrations below the EQS value (7.8 µg/L).

Fig. 4 .

 4 Fig. 4. Zn isotopic compositions for samples from the Egoutier watershed collected along an upstream/downstream profile: (a) Surface waters and WWTP effluents, (b) Total fraction of sediments and road particles, (c) Mobile fraction from sediments and road particles. The location of proven and possible sources of Zn pollution are shown on the upstream/downstream profile (DHC WWTP, oil depot and road traffic). For surface waters and WWTP effluents, the results for the two "grab" sampling campaigns (07-Oct-15, 21-Oct-15) and the integrated sampling with passive DGT (Diffusive Gradients in Thin films) samplers are shown by symbols in various colours. The blue line labelled "GBSW" is the geological background for surface water, which ranges between +0.24‰ and +0.41‰ with a median value of +0.30‰.

Fig. 5 .

 5 Fig. 5. Quantities of AEM (acid-extractable matter, %) and proportion of elements (%) released in the mobile fraction compared to their compositions in the total fraction for sediments and road particles from the Egoutier watershed.

Fig. 6 .

 6 Fig. 6. δ 66/64 Zn versus 1/Zn concentration for surface water and WWTP effluents from the Egoutier watershed obtained by "grab" sampling during two campaigns (07-Oct-15, 21-Oct-15). The solid lines are theoretical mixing lines between a 'geogenic component' (# drilling) and an 'anthropogenic component' represented by WWTP effluents (07-Oct-15). Different percentages for the anthropogenic

Fig. 7 .

 7 Fig. 7. δ 66/64 Zn versus (Al+Fe+Ca)/Zn*10 -4 ) ratio for total fraction of sediments and road particles from the Egoutier watershed. The solid straight line is a theoretical mixing line between a 'geogenic component' (# 1) and an 'anthropogenic component' due to WWTP releases. The δ 66/64 Zn for this contribution is defined by the value of WWTP effluents obtained by DGT (+0.03‰). The dotted line is a mixing line between a 'geogenic component' and an 'anthropogenic component' due to road traffic.

Table Table 1

 Table Concentrations and isotopic compositions of dissolved Zn collected using "grab" sampling and DGT in the Egoutier watershed.

	label	sampling type	date	Zn µg/L	δ 66/64 Zn ±2σ ‰	label	sampling type	date	Zn µg/L	δ 66/64 Zn ‰	±2σ
	Main stream						Groundwater
	#spring	"grab"	18-Feb-15	4	-	-	#drilling "grab"	21-Oct-15	22	0.22	0.01
	#spring	"grab"	09-Apr-15	4	-	-	#drilling "grab"	08-Dec-15	37	-
	#0	"grab"	11-Dec-14	3	-	-	#drilling "grab"	02-Nov-15	21	-	-
	#1	"grab"	18-Feb-15	5	-	-	Tributaries
	#1	"grab"	09-Apr-15	5	-	-	#2-6	"grab"	09-Apr-15	9	-	-
	#3	"grab"	18-Feb-15	35	-	-	#2-6	"grab"	07-Oct-15	18	-	-
	#3	"grab"	10-Apr-15	14	-	-	#2-6	"grab"	21-Oct-15	9	-	-
	#3	"grab"	07-Oct-15	45	-	-	#2-6	"grab"	02-Nov-15	9.1	-	-
	#3	"grab"	21-Oct-15	73	0.10	0.03	#2-6	DGT	07-21-Oct-15	-	0.26	0.05
	#3	"grab"	02-Nov-15	55	-	-	#2-7	"grab"	09-Apr-15	21	-	-
	#3	DGT	07-Oct-15-02-Nov-15	-	0.07	0.03	#2-7	"grab"	21-Oct-15	82	0.12	0.03
	#4	"grab"	18-Feb-15	23	-	-	#2	"grab"	18-Feb-15	51	-	-
	#4	"grab"	10-Apr-15	13	-	-	#2	"grab"	09-Apr-15	44	-	-
	#4-bis	"grab"	10-Apr-15	13	-	-	#2	"grab"	07-Oct-15	35	0.17	0.03
	#4-bis	"grab"	07-Oct-15	30	-	-	#2	"grab"	21-Oct-15	35	0.28	0.02
	#4-bis	"grab"	21-Oct-15	33	0.25	0.02	#2	"grab"	02-Nov-15	29	-	-
	#4-bis	"grab"	02-Nov-15	31	-	-	#2*	DGT	07-Oct-15-02-Nov-15	-	0.13	0.04
	#4-bis	DGT	07-Oct-15-02-Nov-15	-	0.18	0.03	#5	"grab"	18-Feb-15	3	-	-
	#6	"grab"	18-Feb-15	20	-	-	#5	"grab"	10-Apr-15	3	-	-
	#6	"grab"	10-Apr-15	12	-	-	#5	"grab"	07-Oct-15	4.9	-	-
	#6	"grab"	21-Oct-15	27	0.21	0.04	#5	"grab"	21-Oct-15	2	-	-
	#7	"grab"	18-Feb-15	24	-	-	#5	"grab"	02-Nov-15	2.6	-	-
	#7	"grab"	10-Apr-15	17	-	-	#5	DGT	07-Oct-15-02-Nov-15	-	0.27	0.04
	#7	"grab"	21-Oct-15	33	0.17	0.03	Anthropogenic contributions (DHC)
	#8	"grab"	21-Oct-15	28	0.19	0.02 WWTP effluent	"grab"	07-Oct-15	168	0.08	0.01
	#final	"grab"	11-Dec-14	64	-	-	WWTP effluent	"grab"	21-Oct-15	87	0.17	0.04
	#final	"grab"	18-Feb-15	23	-	-	WWTP effluent	DGT	07-21-Oct-15	-	0.03	0.04
	#final #final	"grab" "grab"	10-Apr-15 07-Oct-15	17 27	--	--	Wastewater	"grab"	07-Oct-15	95	0.13	0.01
	#final	"grab"	21-Oct-15	31	0.19	0.02 Wastewater	"grab"	21-Oct-15	78	0.20	0.02
	#final #final	"grab" DGT	02-Nov-15 07-Oct-15-02-Nov-15	35 -	-0.13	-0.03 '-' indicates not analysed

Table 2

 2 Concentrations and isotopic compositions of Zn for sediments and road particles (total and mobile fraction) in the Egoutier watershed.

	sample type	label	sampling	AEM	Zn	δ 66/64 Zn	±2σ
			date	%	mg/kg	‰
	Main stream
	total fraction	#spring	09-Apr-15		96	0.28	0.02
	mobile fraction	#spring	09-Apr-15	4	2.1	0.53	0.02
	total fraction	#1	09-Apr-15		61	0.27	0.02
	mobile fraction	#1	09-Apr-15	3	12	0.40	0.02
	total fraction	#3	10-Apr-15		150	0.13	0.02
	mobile fraction	#3	10-Apr-15	2	54	0.19	0.03
	total fraction	#4-bis	10-Apr-15		51	0.18	0.01
	mobile fraction	#4-bis	10-Apr-15	2	22	0.18	0.02
	total fraction	#final	10-Apr-15		201	0.20	0.02
	mobile fraction	#final	10-Apr-15	16	139	0.22	0.02
	Tributary
	total fraction	#2	09-Apr-15		274	0.08	0.02
	mobile fraction	#2	09-Apr-15	3	174	0.12	0.02
	Road particles
	total fraction	#road particles	14-Nov-15		919	0.16	0.02
	mobile fraction	#road particles	14-Nov-15	16	751	0.19	0.01

Acknowledgments

This work was financially supported by BRGM and the Agence de l'eau Loire-Bretagne. The authors would like to thank mineralogy team for sample preparation, and the ICP-MS team for major and trace-element analyses. Thanks are also due to ISOP2 team project (BRGM and AELB) for helpful discussions. H.M. Kluijver edited the final English version of the paper.

Two anonymous reviewers are thanked for their constructive comments and highlights, which greatly helped to improve the manuscript.

Supplemental online material