Thomas Blanc

Nicolas Goudin

Meriem Mohamad Zaidan

Meriem Garfa Traore

Frank Bienaime

Lisa Turinsky

Serge Garbay

Clément Nguyen

Martine Burtin

Gérard Friedlander

MD Fabiola Terzi
email: fabiola.terzi@inserm.fr

PhD. Marco Pontoglio
email: marco.pontoglio@inserm.fr

THREE-DIMENSIONAL ARCHITECTURE OF NEPHRONS IN THE NORMAL AND CYSTIC KIDNEY

Keywords: Tissue clearing, Kidney, Nephron, Cystic Kidney Disease, Nephronophthisis TRANSLATIONAL STATMENT

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

INTRODUCTION

Kidney maintains body homeostasis via its complex three-dimensional (3D) nephron structure. When this structural organization is altered, renal pathophysiology ensues. Chronic kidney diseases (CKD) are characterized by the development of renal lesions. Intriguingly, pathologists have reported a wide heterogeneity in the distribution of lesions during CKD [START_REF] Rich | A hitherto undescribed vulnerability of the juxtamedullary glomeruli in lipoid nephrosis[END_REF][START_REF] Bankir | Heterogeneity of nephron anatomy[END_REF] .

Whether this reflects the fact that specific nephron segments can be differently damaged, or alternatively, that some nephrons have different susceptibilities to be injured as a whole, is unknown. To address this issue, it is mandatory to reconstruct the 3D shape of nephrons in pathological contexts.

To the best of our knowledge, nephrons have only been fully reconstructed using serial 2D sections [START_REF] Christensen | Threedimensional reconstruction of the rat nephron[END_REF][START_REF] Zhai | Digital threedimensional reconstruction and ultrastructure of the mouse proximal tubule[END_REF][START_REF] Zhai | Three-Dimensional Reconstruction of the Mouse Nephron[END_REF] . Although standard histological sectioning provides high resolution, 3Dreconstructions are very laborious and hard to obtain. This is primarily due to mechanical distortions, which is an inescapable effect of the slicing procedure. Furthermore, 3Dreconstruction from 2D images does not allow direct imaging of 3D structures in wholemounted tissues.

Multiphoton microscopy has improved our ability to detect morphological changes in thick sections. However, a significant limitation is the shallow accessible depth due to lightscattering. By minimizing refractive index differences, clearing agents have dramatically improved the ability to image depths [START_REF] Puelles | We can see clearly now: Optical clearing and kidney morphometrics[END_REF][START_REF] Seo | Clearing and Labeling Techniques for Large-Scale Biological Tissues[END_REF] . Despite the fact that the first clearing agent was introduced a century ago [START_REF] Spalteholz | Uber das Durchsichtigmachen von menschlichen und tierischen Praparaten[END_REF] , it is only recently that several clearing protocols have been developed, mostly for brain [START_REF] Hama | Scale: A chemical approach for fluorescence imaging and reconstruction of transparent mouse brain[END_REF][START_REF] Ertürk | Three-dimensional imaging of solvent-cleared organs using 3DISCO[END_REF][START_REF] Kuwajima | ClearT: a detergentand solvent-free clearing method for neuronal and non-neuronal tissue[END_REF][START_REF] Ke | Optical clearing of fixed brain samples using SeeDB[END_REF][START_REF] Chung | CLARITY for mapping the nervous system[END_REF][START_REF] Tomer | Advanced CLARITY for rapid and highresolution imaging of intact tissues[END_REF][START_REF] Yang | Single-cell phenotyping within transparent intact tissue through whole-body clearing[END_REF][START_REF] Susaki | Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis[END_REF][START_REF] Alanentalo | Tomographic molecular imaging and 3D quantification within adult mouse organs[END_REF] . Recent studies have demonstrated their potential for imaging other solid organs, such as liver, pancreas or kidney [START_REF] Parra | Multiphoton microscopy of cleared mouse organs[END_REF][START_REF] Renier | iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging[END_REF][START_REF] Lee | Improved application of the electrophoretic tissue clearing technology, CLARITY, to intact solid organs including brain, pancreas, liver, kidney, lung, and intestine[END_REF][START_REF] Iversen | Increased glomerular capillary pressure and size mediate glomerulosclerosis in SHR juxtamedullary cortex[END_REF][START_REF] Angelotti | Imaging the kidney: from light to super-resolution microscopy[END_REF][START_REF] Puelles | Validation of a Three-Dimensional Method for Counting and Sizing Podocytes in Whole Glomeruli[END_REF][START_REF] Pannabecker | Role of three-dimensional architecture in the urine concentrating mechanism of the rat renal inner medulla[END_REF][START_REF] Saritas | Optical Clearing in the Kidney Reveals Potassium-Mediated Tubule Remodeling[END_REF][START_REF] Schuh | Combined structural and functional imaging of the kidney reveals major axial differences in proximal tubule endocytosis[END_REF] .

Polycystic kidney disease (PKD), a genetically heterogeneous disorder involving mutations in several ciliary genes, is the most common hereditary kidney disease [START_REF] Braun | Ciliopathies[END_REF][START_REF] Bergmann | Polycystic kidney disease[END_REF] . PKD is characterized by the development of cysts that lead to the complete destruction of the kidney [START_REF] Bergmann | Polycystic kidney disease[END_REF] .

Microdissection studies suggested that cysts can either develop as an "outpushing" of a nephron segment, as in autosomal dominant polycystic kidney disease (ADPKD); an ectatic expansion of collecting ducts, as in autosomal recessive polycystic kidney disease (ARPKD); or exclusively in medullary tubules, as in nephronophthisis (NPHP) [START_REF] Braun | Ciliopathies[END_REF][START_REF] Wilson | Polycystic kidney disease[END_REF] . However, how cysts develop in 3D and organize with each other and normal neighboring tubules is still unknown.

Here, we combined optical clearing with multiphoton microscopy to provide new insights into how nephrons are shaped and organized in physiological conditions and how they are modified during disease, such as cystogenesis. Our results demonstrate that there are three types of nephrons and that vessels may influence nephron spatial organization. Interestingly, we observed that nephrons tend to lie in specific planes. Unexpectedly, when we applied this technique to jck mice, we observed that cysts developed only in specific nephron segments with fusiform cysts intermingled with normal tubules.

RESULTS

Experimental setup

To study the shape of entire nephrons in relation to their environment, we adopted a technique based on optical clearing and multiphoton microscopy (Supplementary Figure S1a). By comparing different optical clearing protocols, we determined that, for kidney, Benzyl Alcohol/Benzyl Benzoate (BABB) was the most appropriate in term of efficacity (Supplementary Figure S1b, S1c and Supplementary Table S1).

3D-reconstruction and digital tracing requires high quality images with high resolution and high signal to background ratio (SBR). We observed that the optical signal provided by native fluorescence was not uniform over kidney sections. In particular, images from the medulla were poorly contrasted with a low SBR (Supplementary Figure S2). In order to improve the SBR, we stained sections with PAS (Supplementary Figure 1d), since we empirically noted that this staining gave rise to high contrast in thin sections during two-photon fluorescence. However, signal was not uniform throughout thick PAS-stained kidney sections (Supplementary Figure 1e). Therefore, we stained kidney sections with fluorochromecoupled lectins: PNA and WGA, which stain glomeruli and tubules, and GSA, which marks blood vessels [START_REF] Laitinen | Changes in the glycosylation pattern during embryonic development of mouse kidney as revealed with lectin conjugates[END_REF] . Remarkably, with the combination of these lectins, SBR was dramatically increased in whole kidney sections with a significant improvement in both the xy-plane and zaxis (Supplementary Figure 1e, Supplementary Movie S1).

3D nephron segment visualization

In order to visualize the shape of whole nephrons, we traced their paths, beginning at the urinary pole of the glomerulus and ending at the collecting duct junction (Supplementary Movie S2). In spite of the fact that the lectins used globally bind all nephron segments, we noticed that when carefully inspected, these lectins were characterized by a specific pattern in the way they stained basal or luminal membranes in the different nephron segments. In other words, we took advantage of the stereotypical pattern of lectin staining to identify the different nephron segments. In this way, we could easily identify six entities: proximal tubule (PT), thin limb (TL) of Henle loop (HL), thick ascending limb (TAL) of HL, distal convoluted tubule (DCT), connecting tubule (CNT) and collecting duct (CD). The transition between PT and TL was characterized by the sudden loss of the typical PT brush border signal and by the decrease of lumen width, which was almost virtually absent in TL (Figure 1a). Conversely, the transition between TL and TAL was characterized by a significant increase of external tubular diameter and relatively constant lumen width (Figure 1b). In all nephrons, the transition of TAL to DCT was systematically found at the vascular pole of the glomerulus. This transition was characterized by a sudden enlargement of external tubular diameter that was predominantly due to a significant increase of the lumen diameter (Figure 1c). The transition between DCT and CNT was characterized by a reduction of both the tubular diameter and lumen (Figure 1d). Finally, the connection between CNT and CCD was characterized by a sudden increase of both tubular diameter and lumen (Figure 1e). The quantification of these morphological transitions was statistically significant (Figure 1f) and its pertinence was verified with the staining with specific tubular markers (Supplementary Figure S3, S4 and S5, and Supplementary Movie S3, S4 and S5).

The shape and size of proximal tubules differ according to their depth

The staining of thick kidney sections allowed us to trace the coordinates of the spatial evolution of entire PT segments. Interestingly, we discovered that their shape was extremely variable and determined by the position of their glomeruli in the cortex. Globally, we identified three patterns. The first corresponded to the most superficial nephrons (SN, Figure 2a, Supplementary Movie S6), which occupied the most external part of the cortex (the most external 30% close to the renal capsule). Their convoluted parts formed compact structures with only 6-7 convolutions around their own glomerulus, occupying very small and tightly packed spaces. The convolution of these SN ended in long and straight pars recta that descended into the medulla. At the opposite end of this spectrum, we observed a second pattern of nephrons located in the deepest part of the cortex (40% more internal depth), next to the medulla (juxtamedullary nephrons, JN, Figure 2a, Supplementary Figure S6, Supplementary Movie S6). Their PT were characterized by an initial short loop that systematically descended toward the papilla and then U-turned to ascend toward the renal capsule. In contrast to SN, JN proximal convoluted tubules were characterized by large coils that evolved around their own glomerulus and occupied large, loosely packed domains in the juxtamedullary cortex. The convoluted part of the PT of JN had 10-15 convolutions, forming large domains. Finally, the third pattern included nephrons located in the middle portion of the cortex (middle nephrons, MN, Figure 2a, Supplementary Movie S6). Interestingly, these tubules had a shape and spatial orientation that represented a transition between SN and JN. In fact, they contained 8-9 convolutions with coils that were larger than SN, but smaller than JN.

In addition, MN had longer pars recta than JN. Interestingly, a global comparison of the shapes of the PT showed that SN and MN had a homogenous pattern whereas JN were extremely heterogeneous (Supplementary Figure S6). In addition, the spatial reconstruction of several nephrons revealed that PT never intermingle (Supplementary Movie S6), indicating that each nephron occupies its own individual space in the cortex.

Morphometric analyses confirmed the large differences in the size and shape of SN and JN, with an intermediate aspect for the MN. PT of JN were more than two-fold longer than the PT of SN (Figure 2b); however, the straightness was greater in SN compared to JN (Figure 2c), consistent with the observation that JN tubules were much more tortuous (Figure 2a, Supplementary Figure S6).

Glomerular size varies with depth

We then analyzed the depth and diameter of glomeruli randomly selected from each of the three zones. Morphometric analyses revealed that glomeruli were located in average 783 and 355 µm from the renal capsule in JN and SN, respectively. As previously reported, we observed that the size of glomeruli varied according to their depth. In particular, the glomerular volume of JN was three-times greater than that of SN and MN (Figure 2d).

3D-nephron reconstruction

We then traced the spatial evolution of entire nephrons from PT to CNT. The global view of their 3D-reconstruction confirmed that nephron shape differed among SN, MN and JN (Figure 3a, Supplementary Movie S7). This difference was mainly due to the structure of the PT. In addition, we also observed that HL had a different 3D spatial organization. In particular, the TL and TAL were more distantly separated in JN than in SN and MN (Figure 3b). Morphometric analyses revealed that JN were globally two-fold longer than SN (Figure 3c). The increased length was proportionally distributed in all segments, except for TAL and DCT which tended to have a similar absolute length in all nephron types (Figure 3d). This suggested that nephron elongation is a surprisingly homogenously patterned process.

The arcuate vessels influence juxtamedullary proximal tubules convolution

To gain a more comprehensive perspective of kidney structure, we took advantage of GSA staining which allowed us to trace the arcuate vessels and their branches into the cortical radiate vessels (Figure 4a and Supplementary Movie S8). Interestingly, the concomitant 3D-reconstruction of nephrons and vessels showed that the arcuate vessels tend to be in a surprising parallel spatial organization with nearby tubules. In particular, we observed that the path followed by specific JN (constrained JN) tends to follow the path of the nearby vessel (Figure 4b, Supplementary Movie S9). Remarkably, this bias (observed only in a subset of JN nephrons) accounted for the large heterogeneity in the shapes of PT (Supplementary Figure S6). Morphometric analyses revealed that the "constrained" PT were significantly longer and more tortuous (reduced straightness) than the "non-constrained" ones (Figure 4c and 4d). It is worth noting that the convoluted parts of both SN and MN were positioned above the arcuate vessels and were not constrained.

Tubules tend to lie in planes

Interestingly, inspection of the 3D shapes of nephrons indicated that they tend to lie in planes (Figure 5a, Supplementary Movie S10). In order to quantify this parameter and to assess the possible statistical bias of this conformation, we measured the general tendency of tubules to bend out of the plane defined by their loops. Our measurements showed that, compared to a simulated model of random walking, generated with the same set of consecutive measured angles and length between consecutive points (Figure 5b), the tubules tended to evolve with smaller deviation from a plane (Figure 5c). This indicated that tubules tended to restrict their path along a plane. The observed differences were statistically highly significant (P < 0.001).

3D-nephron reconstruction reveals a specific pattern for cyst development

To determine whether our protocol allows for tracing nephrons in disorganized pathological tissues, we characterized the shapes and spatial distribution of cysts in jck mice, a widely-used model of nephronophthisis and cystic disease [START_REF] Smith | Development of Polycystic Kidney Disease in Juvenile Cystic Kidney Mice: Insights into Pathogenesis, Ciliary Abnormalities, and Common Features with Human Disease[END_REF][START_REF] Liu | A defect in a novel Nek-family kinase causes cystic kidney disease in the mouse and in zebrafish[END_REF][START_REF] Franke | Magnetic resonance T2 mapping and diffusionweighted imaging for early detection of cystogenesis and response to therapy in a mouse model of polycystic kidney disease[END_REF] . 3D images showed that, in spite of the appearance of prominent cysts, the general 3D course of nephrons did not significantly differ from that of control mice (Supplementary Movie S11). Similarly, morphometric analyses confirmed that the global length of nephrons, glomerular volume and length of each nephron segment were comparable to those of control mice (Supplementary Figure S7). Of note, because of cyst development, we were unable to differentiate TL from TAL in HL. All the nephrons developed fusiform cysts (Supplementary Movie S11 and S12). One of the most striking features was the observation that fusiform cystic dilations occurred in multiple locations along the same nephron segment (Supplementary Movie S12 and S13). Intriguingly, we observed that cysts never developed in PT and HL descending limbs (Figure 6a, Supplementary Movie S12). In contrast, they were predominantly detected in HL ascending limbs, DCT, CNT and in the upper part of CD, in continuity with CNT (Figure 6a, Supplementary Movie S12). In particular, we observed that HL, DCT as well as CNT segments had a peculiarly increased probability to develop cysts in their respective more distal parts (Figure 6b). Quantitative morphometric analyses on 3D-reconstruction images revealed that the total cyst volume per nephron was particularly high in CNT (Figure 7a). In addition, we observed that cyst enlargements in HL and DCT were greater in JN compared to SN and MN (Figure 7a, Supplementary Movie S12). Moreover, cyst incidence was significantly higher in JN compared to the other types of nephrons (Figure 7b). We also observed that the average glomerular inter-distance (calculated on the nearest 5 glomeruli) tended to be particularly increased in cystic mice, especially in the more superficial cortex (Supplementary Figure S8). 3D-reconstruction of numerous cysts revealed that they were extremely variable in shape and volume (Figure 6c, Supplementary Movie S11 and S13). In HL ascending limb, cysts had a fusiform shape with a small diameter. In DCT, cysts had a rather spherical and larger shape and were interspersed among non-dilated tubules. Interestingly, the transition between the DCT and CNT was systematically characterized by a normal non-dilated structure. In contrast, in CNT, we systematically observed a contiguous dilated cystic structure directly in contact with the CD. Interestingly, in CD, this structure progressively shrunk to a normal tubule size. More distally, no further cysts could be detected in the contiguous portion of the CD. Another consistent feature was the peculiar spatial organization of cysts in HL ascending limb. In fact, while cysts were deeper and closer to the loop in SN, they were more superficial and closer to DCT in JN (Figure 6c). Again, cysts occupied an intermediate position in MN (Figure 6c).

DISCUSSION

Traditional histological methods are limited in their ability to detect pathological changes that affect the global shape of nephrons. Here, we present a powerful approach based on tissue clearing that has the potential to address crucial questions in kidney architecture with unprecedented spatial detail in normal and pathological conditions. Our results confirmed the existence of three types of nephrons, which differ in their location, shape and size, consistent with their functional specificities. Moreover, we demonstrated that nephrons tend to lie in planes and to adapt to vessel spatial organization. Intriguingly, when we applied this technique to a model of cystic kidney disease, we observed that cysts develop in all nephrons, but only in specific segments. Interestingly, we showed that cyst shape varies according to the nephron segment, and that along the same nephron, cysts intercalate with normal non-dilated tubules. Altogether, these results provide the first 3D-characterization of the spatial arrangement of nephrons and vessels and importantly provide the basis for understanding a pathological process, such as cystogenesis. Kidney optical clearing is challenging due to high levels of autofluorescence and cell density.

By comparing different clearing protocols, we identified in BABB a powerful technique for implementing the visualization and reconstruction of structures located in the deepest part of the kidney, i.e. the medulla. Moreover, BABB is rapid and scalable and, once cleared, samples can be stored for months before image acquisition. Another main advantage of this technique is its low cost. Clearing agents may result in shrinking or enlargement of structures [START_REF] Hama | Scale: A chemical approach for fluorescence imaging and reconstruction of transparent mouse brain[END_REF][START_REF] Ertürk | Three-dimensional imaging of solvent-cleared organs using 3DISCO[END_REF][START_REF] Kuwajima | ClearT: a detergentand solvent-free clearing method for neuronal and non-neuronal tissue[END_REF][START_REF] Ke | Optical clearing of fixed brain samples using SeeDB[END_REF][START_REF] Chung | CLARITY for mapping the nervous system[END_REF][START_REF] Tomer | Advanced CLARITY for rapid and highresolution imaging of intact tissues[END_REF][START_REF] Yang | Single-cell phenotyping within transparent intact tissue through whole-body clearing[END_REF][START_REF] Susaki | Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis[END_REF][START_REF] Alanentalo | Tomographic molecular imaging and 3D quantification within adult mouse organs[END_REF] . However, since these modifications of kidney size are isotropic, the relative measurements are not expected to be affected or bias their interpretation. One of the most important limitations is the annotation of structures that is highly time-consuming.

Nevertheless, there is a growing number of techniques based on deep learning that should rapidly overcome this limitation.

Consistent with the results obtained by classical techniques, our study showed that nephrons differ in their shape and length according to their position. In particular, we observed that JN have more developed convoluted proximal tubules, larger glomeruli and are two-times longer than SN. The increased length is the result of a harmonious process since lengthening affects proportionally all nephron segments. We also observed that JN have larger HL. Altogether these data clearly show that the microanatomy of SN and JN is significantly different and that the MN display intermediate features. Interestingly, physiological studies have shown that nephrons are also functionally different [START_REF] Bankir | Heterogeneity of nephron anatomy[END_REF] . The morphogenetic events that underlie these differences are not yet known. It is thus tempting to speculate that the particular structure of JN may account for the specificity of their functions.

Interestingly, by providing for the first time the 3D-reconstruction of nephrons and their surrounding vessels, we discovered a spatial constraint between the arcuate vessels and a subgroup of nephrons. Thus, it is conceivable to think that vessels may dictate the way nephrons lengthen during development [START_REF] Barry | Molecular determinants of nephron vascular specialization in the kidney[END_REF][START_REF] Perretta-Tejedor | Endothelial-epithelial communication in polycystic kidney disease: Role of vascular endothelial growth factor signalling[END_REF] . The inspection of the 3D shapes of nephrons combined with computational simulations also revealed that nephrons never intermingle and that each nephron tends to lie in a plane. The functional significance of this observation remains to be elucidated.

Cyst development during PKD is still an intriguing process. NPHP, a pathological condition characterized by cyst development, is the most common genetic disease-causing end-stage renal disease in children and adolescents. Twenty NPHP genes have been identified so far [START_REF] Srivastava | Many Genes-One Disease? Genetics of Nephronophthisis (NPHP) and NPHP-Associated Disorders[END_REF] .

Among them, NEK8 encodes a member of the NIMA (never in mitosis A)-related kinase family which plays a role in cilia function and cell cycle progression [START_REF] Fry | Cell cycle regulation by the NEK family of protein kinases[END_REF] . Nek8 was originally characterized as the gene mutated in jck mice [START_REF] Liu | A defect in a novel Nek-family kinase causes cystic kidney disease in the mouse and in zebrafish[END_REF] . Notably, a mutation in the same protein domain has been shown to lead to NPHP9 in humans [START_REF] Otto | NEK8 mutations affect ciliary and centrosomal localization and may cause nephronophthisis[END_REF] . 2D studies suggested that cyst development dramatically differs among the different forms of PKD [START_REF] Braun | Ciliopathies[END_REF][START_REF] Wilson | Polycystic kidney disease[END_REF] . In particular, in NPHP, cysts seem to be derived exclusively from CD and DCT [START_REF] Smith | Development of Polycystic Kidney Disease in Juvenile Cystic Kidney Mice: Insights into Pathogenesis, Ciliary Abnormalities, and Common Features with Human Disease[END_REF] . Although informative, these immunohistochemical studies cannot argue against the possibility that the loss of specific tubular markers [START_REF] Wilson | Apico-basal polarity in polycystic kidney disease epithelia[END_REF] artificially accounts for this observation. Thus, our 3D study provides the first clear evidence that cyst development is a process that may involve only specific nephron segments. Interestingly, while NEK8 is expressed in the cytoplasm of all nephron segments, its expression in cilia is restricted to DCT and CD. Since these segments are the only prone to develop cysts [START_REF] Smith | Development of Polycystic Kidney Disease in Juvenile Cystic Kidney Mice: Insights into Pathogenesis, Ciliary Abnormalities, and Common Features with Human Disease[END_REF] , we can postulate that the disruption of NEK8 ciliary function is the crucial event for cyst formation. Intriguingly, we also of observed that fusiform cysts are in continuity with normal non-dilated tubules. The fact that a recessive germ-line mutation leads to a pathological phenotype in only a subset of cells suggests that a second event might trigger cyst development in these cells, as suggested for ADPKD [START_REF] Happé | Toxic tubular injury in kidneys from Pkd1-deletion mice accelerates cystogenesis accompanied by dysregulated planar cell polarity and canonical Wnt signaling pathways[END_REF][START_REF] Piontek | A critical[END_REF] .

We also observed that cyst enlargement is more predominant in JN compared to SN, at least considering the cysts that locate between PT and CNT. Consistently, is has been reported that in the context of glomerulosclerosis, glomerular lesions are found more frequently in JN than in SN [START_REF] Rich | A hitherto undescribed vulnerability of the juxtamedullary glomeruli in lipoid nephrosis[END_REF][START_REF] Iversen | Increased glomerular capillary pressure and size mediate glomerulosclerosis in SHR juxtamedullary cortex[END_REF]42 . Whether the overwork due to the increased single nephron glomerular filtration rates and transport/enzymatic activities accounts for the increased susceptibility of JN to deteriorate is an interesting hypothesis that deserves further investigations.

In summary, we describe a new technique to image kidneys in 3D with sufficient molecular specificity and resolution to directly record the spatial and quantitative distribution of specifically labeled internal structures (nephrons, vessels, cysts). Given the convenience, speed and ability for direct quantification, we anticipate that this technique will become a powerful tool for understanding kidney pathophysiology.

MATERIEL AND METHODS

Animals

All animal procedures were approved by the "Services Vétérinaires de la Préfecture de Police de Paris" and by the ethical committee of Université Paris Descartes. Two-month-old FVB/N mice (n=20) were used for setting up the clearing conditions. The study was then conducted on 2-week-old jck male mice (n=4) and control littermates (n=3).

Preparation of renal tissues

Before sacrifice, mice were perfused, via intracardiac catheterization, with 25 ml of heparinized saline (1000 IU/L) followed by 75 ml of 4% paraformaldehyde (PFA) in PBS.

Experiments were performed under xylazine (Rompun 2%; Bayer; 6 μg/g of body weight) and ketamine (Clorketam 1000; Vetoquinol SA; 120 μg/g of body weight) anesthesia.

For clearing studies, kidneys were fixed in 4% PFA for 4 hours, embedded in 4% agarose, and 1.5-mm thick sections surrounding the renal hilum were cut and stored in PBS at 4°C. Kidney sections were first stained and then cleared.

For immunohistochemical studies on thin sections, kidneys were fixed in 4% PFA overnight, paraffin-embedded, and 4-µm sections were cut.

Staining

PAS staining

The 1.5-mm kidney sections were incubated in pure or 1:100 PBS-diluted PAS for 5 minutes at room temperature before clearing. diluted at 1/100 in 0.1% PBS azide and 0.1% Triton-X. Sections were then washed every day for 2 weeks with PBS before clearing.

Immunohistochemistry on thin paraffin-embedded sections

Optical clearing

For Scale clearing, 1.5-mm kidney sections were incubated in ScaleA2 (4M Urea, 0.1% Triton X-100, 10% Glycerol) or ScaleB4 (8M Urea, 0.1% Triton X-100) for 2 weeks, up to 1 year, at 4°C. For BABB clearing, 1.5-mm kidney sections were dehydrated in sequential rinses of EtOH at room temperature. The samples were then incubated in BABB (Sigma-Aldrich)) in a 1:2 ratio for at least 2 days at 4°C.

Two-photon microscopy images acquisition

Tissues were imaged with aqueous gel an inverted multiphoton microscope (Lavision Biotec) equipped with a Mai Tai HP Titanium-Sapphire laser (Spectra-Physics) (Supplementary Figure S1a). The excitation wavelength was 815 nm at 8% of power. We used a 20X water immersion objective (XLUMPLFL20XW, Olympus), N.A. 0.95, W.D. 2.0 mm. For fluorescence acquisition, we used a Non-Descanned Detector at 80% with a bandpass filter 593/40nm. The first step consisted in 2D mosaic image acquisition at the middle z-stack using suboptimal acquisition parameters (400-µm X 400-µm and 1011 X 1011 pixels, with 10% overlap and a line averaging at 2) in order to guide the selection of the most relevant central fields (Supplementary Figure S9a). Once the xy grid and z fields of interest were defined, parameters were adjusted to acquire high quality images. Such approach reduced the region of interest to 5 x 12 fields per z-stack. Then, we acquired 850 z-stacks/optical slices (1-µm step size) surrounding the renal hilum in the middle part of the kidney section.

Image stitching, processing and tracing

Each stack was tiled according to the method developed by Preibisch et al. 43 , which allows the stitching of a large collection of images by using the "Grid/Collection stitching" plugin of the Fiji software (http://fiji.sc/Fiji). Since we observed an important shift between two adjacent images after stitching, we wrote a custom-matching program in Python. This program compensates the image shift allowing us to correctly align the images (Supplementary Figure S9b). Stitched-corrected images were analyzed using Imaris (Version 8.4.2, Bitplane). Tubules and vessels were traced using the filament tracer package manual mode of Imaris and joined by an Imaris Xtension that we developed on Matlab (https://www.dropbox.com/s/sm9u5em3orjrpmc/Standalone_Join_Filament_Tool.zip?dl=0) (Supplementary Figure S9c). Glomeruli and cysts were 3D-reconstructed using the manual mode of the surface package of Imaris. The 3D glomeruli spatial organization was displayed using the manual mode of the spots package of Imaris.

Morphometry

Tubular segment length, nephron length and straightness were determined using the filament tracer package of Imaris. According to the Imaris Software, the straightness was quantified as the ratio between the distance between two points and the length of the nephron segment path. 31 PT and 12 whole nephrons were reconstructed and measured in 3 wild-type mice whereas 37 PT and 17 whole nephrons were reconstructed and measured in 4 jck mice. Cyst and glomerular volumes were determined using the "surface package" of Imaris. 31 and 34 glomeruli were measured in wild-type and jck mice, respectively. 74 cysts were measured in total.

The "out of the plane" angles of tubular turns (indicated as "Beta") were measured between the plane (defined by three consecutive points) and the direction with the next consecutive fourth point in space (Figure 5b). To evaluate the statistical significance of the observed bias, we simulated the distribution of Beta angles with a random rotation of Beta angles (Monte Carlo simulation) in structures that were composed by the same segments with the same set of alpha angles.

For the calculation of the probability to develop a cystic lesion along nephron segments, we represented each nephron by a set of points in space. Each point was annotated in respect for the presence of cyst vs normal structure and the type of segment. The probability at each standardized length (set to 50 bins) was calculated as the ratio between the numbers of points with cystic annotation and the total number of points in the bin.

For the calculation of the average glomerular inter-distance, we considered for each glomerulus the average glomerular inter-distance calculated on the nearest 5 glomeruli.

Data analyses and statistics

Data are expressed as the mean ± S.E.M. Differences between the experimental groups were evaluated using ANOVA followed, when significant (P < 0.05), by the Tukey-Kramer test.

When only two groups were compared, the Mann-Whitney test was used. Statistical analyses were performed using Graph Prism Software. as an example). The degree to which paths tend to go "out of their plane" can be represented by the angle indicated here as "beta". This angle is measured between the segment p3-p4 in respect to the plane (defined by the immediately preceding points p3, p2 and p1) and represented by a dark grey rectangle in the scheme. Calculation of beta angles was then recursively calculated all along the set of points in a tubular path. To evaluate the extent of the means ± S.E.M. Analysis of variance (ANOVA) followed by Tukey-Kramer test: JN versus SN: # P < 0.05; JN versus MN: * P < 0.05, ** P < 0.01. A valuable strategy for visualizing the complex structure of kidneys at nephron level in physiological and pathological conditions.

4 -

 4 µm sections of paraffin-embedded kidneys were heated for antigen retrieval and incubated overnight at 4°C with fluorochrome-coupled lectins in order to trace the tubules (Rhodamine Peanut Agglutinin [PNA; RL-1072-5] and Rhodamine Wheat Germ Agglutinin [WGA; RL-1022-5], Vector Laboratories, diluted at 1/200) and segment-specific primary antibody in order to depict distinct tubular segments (Biotinylated Lotus Tetragonolobus Lectin [LTL; B-1325], Vector Laboratories, diluted at 1/1 00; mouse anti-Calbindin D28K [D-4], Santa Cruz, diluted at 1/200; goat anti-AQP2 [C-17], Santa Cruz, diluted at 1/200). The next day, the sections were incubated with the secondary antibody for 1 h at room temperature (Alexa Fluor 488 Conjugate, [S32354], Invitrogen; anti-Goat/Alexa Fluor 488 [A-11055], Invitrogen; anti-Mouse/Alexa Fluor 488 [A-21202], Invitrogen; all diluted at 1/500) before being colored with DAPI. All images were acquired with a Nikon Eclipse E800 Microscope and prepared using FiJi Software. Lectin staining on thick sections The 1.5-mm thick kidney sections were incubated at 4°C for 1 month in Texas Red or Fluorescein-Isothiocyanate-coupled lectins: PNA [RL-1072-5] and WGA [RL-1022-5],

  developmental switch defines the kinetics of kidney cyst formation after loss of Pkd1. Nat Med. 2007;13(12):1490-1495. 42. Ikoma M, Yoshioka T, Ichikawa I, Fogo A. Mechanism of the unique susceptibility of deep cortical glomeruli of maturing kidneys to severe focal glomerular sclerosis. Pediatr Res. 1990;28(3):270-276. 43. Preibisch S, Saalfeld S, Tomancak P. Globally optimal stitching of tiled 3D microscopic image acquisitions. Bioinformatics. 2009;25(11):1463-1465.

FIGURE LEGEND Figure 1 :

 LEGEND1 FIGURE LEGEND

Figure 2 :

 2 Figure 2: 3D proximal tubule reconstruction reveals three different shapes and

Figure 3 :

 3 Figure 3: 3D nephron reconstruction reveals three different types of nephrons. (a)

Figure 4 :

 4 Figure 4: Vessels determine nephron shape. (a) 3D reconstruction of vessel architecture

Figure 5 :

 5 Figure 5: Nephron tubules tend to evolve as a flat structure. (a) A typical path of a

  Figure 1

ACKNOWLEDGMENTS

We thank the LEAT, Histology and Imaging Platforms of Structure Federative de Recherche Necker for technical assistance. We thank Pierre Isnard, Marie-Claire Gubler and Nicolas Kuperwasser for critical reading of the manuscript. This work was supported by Institut National de la Santé et de la Recherche Médicale, Université Paris Descartes, Assistance Publique Hôpitaux de Paris, Agence Nationale Recherche, Whoami Laboratoire d'Excellence, Pharma Research and Early Development Roche Laboratories (Basel, Switzerland) and Institut Roche de Recherche et Médecine Translationnelle (Paris, France).

DISCLOSURE STATEMENT

The authors declare that they have no conflict of interest.

AUTHOR CONTRIBUTIONS


M Louzon

B Pauget

A Pelfrêne

F Gimbert

A De Vaufleury
email: annette.devaufleury@univ-fcomte.fr

Combining human and snail indicators for an integrative risk assessment of metal(loid)contaminated soils

Keywords: bioavailability, bioaccessibility, metal(loid)s, hazard quotient, carcinogenic risk Cantareus aspersus

With the new soil uses such as land restoration and to protect wilderness, the human health risk assessment (HHRA) and environmental risk assessment (ERA) should be combined. Based on the relationships demonstrated between an indicator of soil quality, the land snail, and human exposure, the aim of this study is to examine the snail and human risk indicators twenty-nine soils contaminated by metal(loid)s. HHRA was evaluated by both hazard quotient and carcinogenic risk. When the human health indicators were ranked as uncertain, they were weighted by bioaccessibility to refine the risk assessment. The ERA was performed with risk coefficient after ex situ snail exposure. The results showed strong and novel relationships between human health and environmental risk indicators that had never been found before. For 62 % of the soils, both indicators revealed either a confirmed risk or an uncertain level of risk. Overall pollutants present greater risk for human than for environment, with 55 vs 28 % of the studied soils classified in the proven risk, respectively.

An original integrative risk assessment of polluted soils has been proposed, that shall help setting up relevant strategies to manage contaminated soils considering not only human but also environmental indicators of risk.

Introduction

The One Health concept demonstrates that human, animal and ecosystem health are interrelated, notably concerning infectious diseases and chemical contamination [START_REF] Aguirre | One health -Transdiciplinary opportunities for SETAC leadership in integrating and improving the health of people, animals and the environment[END_REF][START_REF] Lebov | A framework for One Health research[END_REF][START_REF] Destoumieux-Garzon | The One Health Concept: 10 years old and a long road ahead[END_REF][START_REF] Harrison | EcoHealth and One Health: A theory-focused review in response to calls for convergence[END_REF]. This concept is not currently applied to the management of contaminated soils in France, in verifying the compatibility of soil uses (e.g., community gardens, recreational areas), or in assessing the environmental risk [START_REF] Meem | Méthodologie Nationale de Gestion des Sites et Sols Pollués. Direction générale de la prévention des risques[END_REF]. Moreover, the remediation of industrial and urban wastelands requires the assessment of both the human and ecological health risks in the context of increasing alternative uses to both limit urban sprawl and protect the wilderness (such as through land restoration approaches). In most cases, only the total concentration of contaminants in soil is considered [START_REF] Jia | Assessment of soil heavy metals for eco-environment and human health in a rapidly urbanization area of the upper Yangtze Basin[END_REF], although according to the bioavailability concept, only a fraction of these contaminants is available and may therefore be assimilated by organisms, leading to adverse effects [START_REF] Allan | Bioavailability of metals in terrestrial ecosystems: importance of partitioning for bioavailability to invertebrates, microbes and plants[END_REF]ISO 17402, 2008;ISO 19204, 2017).

To determine an accurate assessment of risk, there is a real need to refine human health and environmental risk assessments (HHRA and ERA, respectively) to account for the bioavailable concentrations of contaminants and to combine these assessments to propose an integrative risk assessment (IRA) of contaminated soils [START_REF] Alvarenga | Chapter 3 -Indicators for monitoring mine site rehabilitation[END_REF]Louzon et al., 2020a). This IRA aims associating HHRA and ERA in the frame of the One health concept applied to the management of terrestrial pollutions. Nowadays, HHRA is very often the driving endpoint in the risk assessment of contaminated soils, while the ERA is not systematically carried out in the current methodology for the management of these soils. Yet, usually HHRA and ERA are in most of the cases studied separately. In France, the method to assess the compatibility of the toxicological risks with the current uses of the site, that is called "the interpreting the state of the environment (ISE)" may be used to perform the HHRA [START_REF] Meem | Méthodologie Nationale de Gestion des Sites et Sols Pollués. Direction générale de la prévention des risques[END_REF]. These toxicological risks are assessed by the comparison of hazard quotients (HQ) and carcinogenic risk (CR) to toxicological reference values (TRV). To refine the method of exposure and risk assessment (especially when the values of HQ and CR showed uncertain levels of risk), the bioaccessible concentrations of pollutants measured in soils according to validated and standardized protocols may be used [START_REF] Pelfrêne | Assessment of potential health risk for inhabitants living near a former lead smelter. Part 2: sitespecific human health risk assessment of Cd and Pb contamination in kitchen gardens[END_REF][START_REF] Meem | Méthodologie Nationale de Gestion des Sites et Sols Pollués. Direction générale de la prévention des risques[END_REF]ISO 17924, 2018). In this case, total soil concentrations are weighted by the bioaccessible fraction determined using the UBM (unified bioaccessibility method) test [START_REF] Meem | Méthodologie Nationale de Gestion des Sites et Sols Pollués. Direction générale de la prévention des risques[END_REF]. Some terrestrial bioassays and bioindicators can be utilized to perform an ERA using for example the risk coefficient (RC) based on the identification of excess transfers and toxicity of contaminants to the land snail [START_REF] Pauget | The SET and ERITME indices: Integrative tools for the management of polluted sites[END_REF]Louzon et al., 2020b). As previously determined in soils contaminated by As, Cd and Pb, strong relationships exist between oral bioaccessibility to humans and bioavailability to snails (Louzon et al., 2020a), making these methodologies relevant candidates to perform an IRA for these metal(loid)s. The IRA methodology requires established amplitudes of the responses of indicators used and the convergences and/or divergences between them. The goals of this study were to examine indicators of human health and environmental risks of contaminated soils with using the land snail, Cantareus aspersus, as a bioindicator of soil quality (de [START_REF] De Vaufleury | Landsnail for ecotoxicological assessment of chemicals and soil contamination[END_REF]Itziou et al., 2018;[START_REF] Carbone | Helix aspersa as sentinel of development damage for biomonitoring purpose: A validation study[END_REF][START_REF] Baroudi | Snail as sentinel organism for monitoring the environmental pollution; a review[END_REF][START_REF] Radwan | Biomarker responses in terrestrial gastropods exposed to pollutants: A comprehensive review[END_REF], to evaluate their complementarity and propose a way to combine them to perform IRA of contaminated soils. This novel evaluation was conducted in accordance with the French ISE methodology (MEEM, 2017), which is based on comparisons of the state of the environment (i.e., the risk of soil contamination) and the current anthropic uses (HHRA) and the transfers to organisms of the soil fauna (ERA).

Experimental

Soils

Twenty-nine soils contaminated with different mixtures and concentrations of As, Cd and Pb were sampled (0-25 cm deep) in France after humus removal (Table 1). Next, these soils were dried (< 40°C) and sieved through 250 µm. The physicochemical parameters of these soils (clay, silt, sand, organic matter (OM), carbonate (CaCO3) contents, pHwater, cationic exchange capacity (CEC), and the oxidizable cations of aluminium (Alox) and iron (Feox)) were previously characterized by Louzon et al. (2020a) and were summarized in table S1. The minimal and maximal values of the physicochemical parameters were 4.3 and 8.3 for pH water, 4.3 cmol kg -1 and 38.8 cmol kg -1 for CEC, 0.020 cmol kg -1 and 0.054 cmol kg -1 for Alox, 0.005 cmol kg -1 and 0.008 cmol kg -1 for Feox, < 1.00 g kg -1 and 169 g kg -1 for CaCO3 content, 15.4 g kg -1 and 360 g kg -1 for OM content, 68.0 g kg -1 and 416 g kg -1 for clays, 75.0 g kg -1 and 734 g kg -1 for silts and 66.0 g kg -1 and 837 g kg -1 for sands. The degree of soil contamination (Table 1) were previously characterized in Louzon et al. (2020a), and the min and max concentrations were 3.28 mg kg -1 dry weight (dw) and 228 mg kg -1 dw for As, 0.10 mg kg -1 dw and 257 mg kg -1 dw for Cd and 17.8 mg kg -1 dw and 8971 mg kg -1 dw for Pb.

Human health risk assessment

HHRA was performed in accordance with the French methodology (MEEM, 2017).

On the basis of the concentration of each of the metal(loid)s in the soils, the average daily dose ((ADD) in mg kg -1 of body weight day -1 ) was calculated according to the ingestion of soil particles [START_REF] Liang | Heavy metal contamination and health risk assessment in the vicinity of a tailing pond in Guangdong, China[END_REF][START_REF] Jia | Assessment of soil heavy metals for eco-environment and human health in a rapidly urbanization area of the upper Yangtze Basin[END_REF] with generic input data from the ISE calculation grid (MEDD, 2007a). More precisely, the data considered that 91 mg of soil [START_REF] Stanek | Soil ingestion distributions for Monte Carlo risk assessment in children[END_REF][START_REF] Meem | Méthodologie Nationale de Gestion des Sites et Sols Pollués. Direction générale de la prévention des risques[END_REF] could consumed daily by a child (15 kg) over 340 days for 6 years of theoretical exposure (Eq. 1).

= × × × × × 10 (1)
where: CS: concentration of contaminant in soil (mg kg -1 ) IR: ingestion rate of soil (mg day -1 ) EF: exposure frequency (day year -1 ) ED: exposure duration (years) BW: body weight (kg) AT: averaging time (days) for non-carcinogenic contaminants: ED (years) x 365 days year -1 [START_REF] Bevan | Threshold and non-threshold chemical carcinogens: A survey of the present regulatory landscape[END_REF] or for carcinogenic contaminants: 70 years (i.e., lifetime) x 365 days year -1 .

With ADD, both the threshold effects by calculating the hazard quotient (HQ) and the nonthreshold effects by calculating the carcinogenic risk (CR) were considered by using the toxicological reference values (TRV) of each contaminant (Table 2) recommended by ANSES (2020) and INERIS (2018) (Eqs. 2 and 3) (MEDD, 2007b;[START_REF] Liang | Heavy metal contamination and health risk assessment in the vicinity of a tailing pond in Guangdong, China[END_REF][START_REF] Jia | Assessment of soil heavy metals for eco-environment and human health in a rapidly urbanization area of the upper Yangtze Basin[END_REF], where TRV is expressed in mg kg -1 day -1 for threshold chemicals and in (mg kg -1 day -1 ) -1 for carcinogens.

=

(2)

= ×

(3)

The interpretation of HQ and CR for As, Cd and Pb was ranked into three levels: no risk, uncertain risk and potential risk (Table 3). When values of HQ and/or CR are uncertain (Table 3), ADD can be weighted by the percentage of bioaccessible fraction measured with UBM (ISO 17924, 2018;Louzon et al., 2020a). The bioaccessible fractions (expressed in %)

of the metal(loid)s corresponded to the ratio between the measured concentrations and soil concentrations (expressed in mg kg -1 dw). To refine the exposure assessment, bioaccessibility can be introduced into HHRA by using a relative bioavailability factor (RB) to quantify the difference in bioavailability of a metal(loid) between the soil matrix and the reference matrix (on which the TRV was built). This factor can be then used in the calculation of ADD (Eq. 4).

!" = × # = × $%&' ()* = × $%&' ×+ $%&' ()* ×+ ()* (4)
where RBsoil and RBTRV are the absolute bioavailability of the metal(loid) in the soil matrix and the reference matrix, respectively (unitless); BAsoil and BATRV is the absolute bioaccessibility of the metal(loid) in the soil matrix and the reference matrix, respectively (unitless); fasoil and faTRV are the absorbed fraction of the metal(loid) extracted from the soil matrix and the reference matrix, respectively (unitless). Based on INVS (2012), it was found that for As and Cd: (i) fasoil and faTRV were similar, and (ii) the absolute bioaccessibility in the gastric (G) phase (i.e., stomachal phase) of these elements in drinking water (i.e., faTRV) was close to 100 %. Thus, for As and Cd, the bioaccessible fraction was directly used to weight the ADD (Eq. 5).

!" , -./ 01 = × # 234 (5)

A similar approach was used for Pb. The TRV established by WHO was calculated considering all sources of Pb (water, air, and food) with the aim of not exceeding a blood level of 50 µg L -1 in a child. The calculation was performed on the basis of: (i) a Pb uptake of 20 to 80 % in children's diets [START_REF] Ziegler | Absorption and retention of lead by infants[END_REF][START_REF] De Michele | Nutrition of lead[END_REF] and (ii) a Pb retention of 30 % in the body. According to [START_REF] Oomen | How can information on oral bioavailability improve human health risk assessment for lead-contaminated soils? Implementation and scientific basis[END_REF], the absolute bioavailability of Pb in the reference matrix can be estimated to average 40 %. Thus, the adjusted ADD for Pb can be determined by considering the maximum value of the absorption (80 %) for all soils (Eq. 6) [START_REF] Invs | Guide pratique. Quantités de terre et poussières ingérées par un enfant de moins de 6 ans et bioaccessibilité des polluants : état des connaissances et propositions[END_REF].

5067-890 ,

:;1 = × $%&' ×<.> <.? = × 2 × # 234 (6)
For a conservative estimate, the phase (gastric or gastro-intestinal, i.e., G or GI) in which oral bioaccessibility is the highest was used to weight the ADD [START_REF] Invs | Guide pratique. Quantités de terre et poussières ingérées par un enfant de moins de 6 ans et bioaccessibilité des polluants : état des connaissances et propositions[END_REF]. Finally, the ISE was performed for each soil to obtain the HHRA by considering the worst interpretation of risk indicators for all contaminants (e.g., if HQ and CR for As and Pb rank a soil as no risk, but its HQ for Cd rank the soil as proven risk, the soil is then considered to have risk due to the Cd) using the criteria summarized in table 3.

Environmental risk assessment

The RCs for environmental risk assessments were obtained using the snail ex situ test over 28 days (Louzon et al., 2020b). Sub-adult snails (Cantareus aspersus) were exposed as described in Louzon et al. (2020a). To assess environmental risk by RC calculation based on bioaccumulated concentrations, the following formula was used with the toxicity point (TP)

from ATSDR ( 2015) and ex situ threshold guide values (TGV) according to Louzon et al. (2020b) and table 2 (Eq. 7).

= AB C" 3 D E2DE"D!F !32D 2+ E2D! G3D D! 3D D 34 H3 E"F ! I> J K L -1N × : (7)
The ERA was performed for each soil by the evaluation of the risk of transferred metallic elements (ERITME) index based on the sum of the RCs (Pauget and de Vaufleury, 2015) (Eq.

8).

The interpretation of the level of risk as a function of RC and/or ERITME values is possible on the basis of ecotoxic effects on the kinetics of sexual maturity (Louzon et al., submitted) (Table 3).

O P QO = RC + RC + RC UV (8)

Statistical analysis

The statistical analysis was performed with R (version 3.4.2) (R Core Team, 2018). The assessment of the relationships between the indicators of human health and environmental risks were performed using Spearman's correlation. The relationships between the total concentrations of metal(loid)s in the soils and the excess metal(loid) transfers to snails (C28 days > TGV) were evaluated by comparisons of the identity line (y = x) of the ratio between the total concentration in the soils and the SGV (soil guide values) [START_REF] Pauget | Ranking field site management priorities according to their metal transfer to snails[END_REF] as a function of the ratio between the internal concentration in the snails after 28 days of exposure and the TGV.

Results and discussion

Human health risk assessment

HQ and CR were calculated for each soil to estimate human health risk (Table 4). By considering the total concentrations of metal(loid)s in the twenty-nine soil samples, the HQ ranged from 0.04 to 2.86 for As, from 0 to 4.04 for Cd and from 0.16 to 80.5 for Pb, respectively. The CR ranged from 2.38 x 10 -6 to 1.65 x 10 -4 for As and 7.33 x 10 -8 to 3.69 x 10 -5 for Pb. Overall, for the HHRA, the HQ and CR indicators demonstrated divergence in several cases for the same metal(loid)s. Values of these indicators were uncertain for 16, 9

and 16 soils for the HQ of As, Cd and Pb, respectively, and 27 and 20 soils for the CR of As and Pb, respectively (in table 3, the uncertainty area concerns values between 0.2 to 5 for HQ and 10 -6 to 10 -4 for CR).

According to [START_REF] Invs | Guide pratique. Quantités de terre et poussières ingérées par un enfant de moins de 6 ans et bioaccessibilité des polluants : état des connaissances et propositions[END_REF], to refine the risk assessment, the HQ adjusted and the CR adjusted were calculated on the basis of the highest bioaccessible fraction obtained between the G and the GI phases (Table 4). The adjustment of ADD for Pb can be made by considering the maximum value of the absorption (80 %) and the mean bioavailability (40 %).

For As, the bioaccessible fractions were higher in the GI phase than in the G phase for 9 soils (O1, O12, O13, O14, O16, O18, O19, O20 and O22), whereas for Cd and Pb, the bioaccessible fraction was systematically higher in the G phase than in the GI phase (Table 1). For these metals, the higher bioaccessibility in the G phase is explained by the acidic pH of the stomach-simulated fluids, which contributes to higher solubilization of metals [START_REF] Ruby | Estimation of lead and arsenic bioavailability using a physiologically based extraction test[END_REF][START_REF] Wragg | An inter-laboratory trial of the unified BARGE bioaccessibility method for arsenic, cadmium and lead in soil[END_REF]. For As, the relatively similar level of bioaccessibility between the G and GI phases could be explained by the reduced adsorption and precipitation reactions caused by the particularly geochemical behaviour of this metalloid in the simulated fluid conditions at a neutral pH [START_REF] Ruby | Estimation of lead and arsenic bioavailability using a physiologically based extraction test[END_REF][START_REF] Wragg | An inter-laboratory trial of the unified BARGE bioaccessibility method for arsenic, cadmium and lead in soil[END_REF][START_REF] Rahman | Arsenic bio-accessibility and bioaccumulation in aged pesticide contaminated soils: A multiline investigation to understand environmental risk[END_REF].

For As, after adjustments of the values in the uncertainty area, the HQ and CR adjusted ranged from 0.02 to 2.86 and 4.28 x 10 -7 to 1.65 x 10 -4 , respectively, leading to decreased numbers of soils (7 and 23, respectively) with uncertain risk (Table 4). In the current management of As polluted soils CR is shown to be present in very low concentrations in soil, notably at the SGV of As in soils (25 mg kg -1 ). Indeed, the soil O7 for example, presented a measured concentration of As of 12.8 mg kg -1 (Table 1), and the unadjusted and adjusted CRs were 9.27 x 10 -6 and 3.24 x 10 -6 , respectively (Table 4), despite the low bioaccessible fractions (35 % and 25 %, respectively for the G and the GI phases). In this example, the adjustment of the CR by bioaccessibility is not sufficient to reduce the uncertainty of the risk to the level of "no risk" (Table 4). The adjustment of the HQ in the area of uncertain risk for

Cd by bioaccessibility provides a HQ ranging from 0 to 4.04 with a decrease of 3 % in the number of soils with uncertain risk (i.e., 31 % and 28 % of the soils with uncertain risk for HQ and HQ adj., respectively) (Table 4). The decrease in the number of soils with uncertain risk for the HQ of As was higher than for Cd in relation to the lower gastric bioaccessibility of As versus Cd in the soils of this study (G bioaccessible median fraction for As and Cd of 32 % and 84 %, respectively) (Table 1). For As, and to a lesser extent for Cd, these results validated the inclusion of bioaccessibility to improve the interpretation of human health risks, both in terms of toxicological threshold effects and carcinogenic effects. For Pb, the adjusted HQ and CR ranged from 0.16 to 80.5 and from 7.33 x 10 -8 to 7.38 x 10 -5 , respectively (Table 4). However, due to the correction applied to obtain a better ADD consideration for Pb, the use of an orally bioaccessible fraction to refine the HQ or CR of Pb seems inefficient, unless the bioaccessible fraction was smaller than 50 % (see eq. 6 in 2.2. Materials and methods section).

The results of the ISE showed that none of the studied soils presented no risk to human health, notably due to the contamination of these soils by Pb. However, after the soils with uncertain risk were ranked based on adjustments of HQ and CR, two soils became no risk (O16 and O22). For an ISE based on results that were risk adjusted or non-risk adjusted with bioaccessibility, the results showed that 11 and 18 soils, respectively, had uncertain risk, leading to a soil status modification of 24 % (Table 4). The relevance of adjusting HQ and CR with the bioaccessible fraction to decrease the uncertainty regarding the risks of contaminated soils (carcinogenic or not) to human health was observed, particularly for soil contaminated by As (Table 4). However, because Pb and its adjustment with eq. 6 mainly drive the ISE, the bioaccessibility fraction in this study was not sufficient to decrease the global risk for HHRA for most of the soils. The use of the bioaccessible G fraction is questionable for physiological reasons. Indeed, metal(loid) absorption mainly occurs in the intestine [START_REF] Mushak | Gastro-intestinal absorption of lead in children and adults: Overview of biological and biophysico-chemical aspects[END_REF]Diamond et al., 1997). Currently, in the regulations (INVS, 2012), the most penalizing phase (often the G phase) is considered. Consequently, by considering the G phase, which is in the most of the cases, the most penalizing to be more protective and decrease the uncertainty regarding risk, the risk can be overestimated. Due to the consideration of the absorption abilities of Pb in the human digestive tract (eq. 5), the oral bioaccessibility fraction improved the HHRA and demonstrated human health risks in 5 additional soils (Table 4). However, the G phase is the main phase considered to decrease the uncertainty caused by Pb in relation to the stronger bioaccessibility of Pb in the stomach (Table 1). For physiological reasons, if the gastro-intestinal phase is considered (i.e., the main site of contaminant assimilation), the results could be different. Indeed, the median (min, max) of the bioaccessible fractions of Pb in the gastric and gastrointestinal phases was 84 % (17 %, 100 %) and 5 % (0 %, 45 %), respectively (Table 1).

Environmental risk assessment

The snail ex situ test was used to assess the environmental risk of these contaminated soils by measuring the bioaccumulated concentrations of metal(loid)s in the visceral mass (Table 1). The results showed that the concentrations of metal(loid)s ranged from 0.098 to 21.9 mg kg -1 of dw viscera for As, from 2.53 to 333 mg kg -1 for Cd and from 1.42 to 856 mg kg -1 for Pb. An inter-soil variability of bioaccumulated concentrations in C. aspersus was found and this variability reflected the influence of the soil contamination (concentration of metal(loid)s in the soils and mixtures) and the influence of the physicochemical parameters of the soils on metal(loid) mobility and transfer, such as the silt content, the cationic exchange capacity and the organic matter content [START_REF] Pauget | Soil parameters are key factors to predict metal bioavailability to snails based on chemical extractant data[END_REF]Louzon et al., 2020a). On the basis of the ex situ threshold guide values (TGV), RCs were calculated for each soil and metal(loid) and summed for the assessment of risk by the ERITME index calculation (Table 5). RCs ranged from 0 to 35500 for As, 0 to 23400 for Cd and 0 to 37400 for Pb. ERITME for the three metal(loid)s studied varied from 0 to 96300 (Table 3). Finally, according to the bioavailability of As, Cd and Pb for the land snail, 11, 10 and 8 soils were classified as without risks, with uncertain risk and with proven environmental risk, respectively (Table 5).

The relationships between the ratio of the concentrations of metal(loids) in the soils with the soil guide values (SGV) and the ratio of the concentration of metal(loid)s in the viscera of snails after 28 days of exposure to the threshold guide value (when the ratios between the internal concentrations and TGV are higher than 1, a bioavailability of contaminant is characterized) are shown in figure 1. Soils below the identity line (y = x) showed a high bioavailability of metal(loids)s in snails based on internal concentrations of the metal(loid)s measured in the snails, while soils above the line showed low bioavailability of excess metal(loids)s in the soils to snails (Figure 1). This inter-soil variability according to the total concentrations in soils and in the snail is related to the concept of the bioavailability. More precisely, for As transfer to snails, this variability results from the positive influence of silt and the negative influence of organic matter content in soils (Louzon et al., 2020a). The values of RCs and the ERITME index constituted a basis to prioritize the management of polluted soils (e.g., remediation, adjustment of use) according to the environmental risk on the basis of the toxicity of metal(loid)s transferred to snails [START_REF] Pauget | The SET and ERITME indices: Integrative tools for the management of polluted sites[END_REF][START_REF] Mariet | Using bioindicators to assess the environmental risk of past mining activities in the Vosges Mountains (France)[END_REF]Louzon et al., 2020b). For example, by considering the ERITME values for soils O28 and O7 (724 and 12328, respectively), the O7 soil presented a higher environmental risk than O28, and its management should be prioritized to limit environmental risk, although the soil O7 was less contaminated (Table 5). Considering the variability of the values of RC that reflect the soil contamination and their physico-chemical properties (Tables 1 and5), the bioavailability assessment is a way to refine environmental impact assessments according to the specificity of each site in terms of physicochemical parameters [START_REF] Bradham | Effect of soil properties on lead bioavailability and toxicity to earthworms[END_REF][START_REF] Pauget | Ranking field site management priorities according to their metal transfer to snails[END_REF].

Correlations between human health and environmental risk indicators

Spearman's correlation coefficients (ρ) were calculated between human health risk indicators (HQ or CR) either adjusted or not adjusted for bioaccessibility if the values were in the area of uncertain risk and the environmental risk indicator assessed with land snails (RC), and are summarized in table 6. Strong correlations were demonstrated between RC and values of human health risk indicators with significant ρ between 0.57 to 0.93. For HQ, the adjustment with bioaccessibility increased the correlations with RC for Pb (+2 %) but not for Cd, and it decreased the correlations with RC for As (-10 %). For CR, the adjustment with bioaccessibility increased the correlations with RC for As (+13 %) and for Pb (+3 %) (Table 6). Overall, the correlations between human health and environmental risk indicators were improved by considering oral bioaccessibility for humans. On the basis of the correlations obtained, the relationships discovered between oral bioaccessibility and the bioavailability of metal(loid)s in soils as bioindicators of soil quality by [START_REF] Rahman | Arsenic bio-accessibility and bioaccumulation in aged pesticide contaminated soils: A multiline investigation to understand environmental risk[END_REF] and Louzon et al. (2020a) can be extended between human health (non-carcinogenic and carcinogenic) and environmental health risk indicators.

Integrative risk assessment

According to the strong correlations observed, the interpretation of the assessment of the human health, whether adjusted or not adjusted with oral bioaccessible fraction and environmental risk was compared for each soil to evaluate similarities and discrepancies (Figure 2). In figure 2, on the basis of the values used to interpret the indicators of risk (Table 3), the worst level of human health risk with HQ and/or CR was used to rank the soils.

The comparison between HHRA or HHRA adj. and ERA showed that 52 % and 45 % of the soils, respectively, had the same level of risks (Figure 2). These differences can be related to the parameters used to consider the absorption of Pb (see eq. 6 in subsection 2.2. of the Materials and methods) that increases the values of HQ and CR adjusted. For example, without adjustment, the HQ for Pb of soil O1 was 3.86 (uncertain risk), while with adjustment, the HQ was 6.25 (proven risk) (Table 4).

These results demonstrated convergences and some differences in terms of interpretation between the conclusions of the ERA and the HHRA of the soils. For example, existing human activities at the site of soil O28 appear inappropriate due to the state and the fate of the soil contamination, while this does not seem to be the case for the soil fauna organisms. Indeed, the RC of Pb in the O28 and O14 soils differed greatly (724 and 11560, respectively) with various environmental risk interpretations (no risk and uncertain risk) (Table 4), while their total soil concentrations were higher (467 mg kg -1 and 719 mg kg -1 ) (Table 1). Their human health risk was also higher (HQ for Pb of 4.19 and 6.45 for O28 and O14 soils, respectively).

The HQ of the Pb in these soils was close to 5, and classifying the soils as having high uncertain and proven risks, respectively (Table 4). This example illustrates the different conclusions that can be drawn between HHRA and ERA. For HHRA, these two soils are classified as high uncertain and proven risk, respectively, which is in agreement with the alert threshold of saturnism risk (300 mg kg -1 ) and its vigilance threshold (100 mg kg -1 ) (HCSP, 2014), which implies management of soil pollution (notably by considering the G phase). For soil fauna, soils O14 and O28 were classified as either having uncertain risk and no risk, respectively.

The overall assessment of the environmental risk cannot be performed only on the basis of the response of one bioindicator. However, the use of an accumulation bioindicator that shows the transfer and the bioavailability of contaminants provides essential data regarding risk. This information constitutes the necessary elements of decisions-making to help manage the pollution of the contaminated soils. The IRA was performed for each soil by considering the worst interpretation of the risk to be the most protective for the health of the ecosystem including humans (One Health). Finally, of the twenty-nine investigated soils, often highly contaminated with As, Cd and Pb (in terms of mixture and concentration), fifteen soils presented risks and fourteen were of uncertain risk (Figure 2). For these last fourteen soils, supplementary investigations are required to refine the level of risk, potentially by using others bioindicators such as earthworms [START_REF] Fründ | Earthworms as bioindicators of soil quality[END_REF] and specific assays, to assess for example the carcinogenic [START_REF] Vasseur | Ecogenotoxicology in earthworms: A review[END_REF]de Souza et al., 2015;[START_REF] Liu | Assessment of the mutagenicity of sediments from Yangtze river estuary using Salmonella typhimurium/microcosme assay[END_REF] and endocrine disruptor [START_REF] Höss | Endocrine disruption in nematodes: effects and mechanisms[END_REF][START_REF] Mertl | Characterization of estrogen and androgen activity of food contact materials by different in vitro bioassays (YES, YAS, ERα and AR CALUX) and chromatographic analysis (GC-MS, HPLC-MS)[END_REF][START_REF] Druart | A full life-cycle bioassay with Cantareus aspersus shows reproductive effects of a glyphosate-based herbicide suggesting potential endocrine disruption[END_REF] potentials of these soils (ISO 19204, 2017). These approaches that would possibly involve integrated biomarker responses (IBR) can be coupled with the ERITME index as demonstrated by [START_REF] Mleiki | Integrative assessment of the biological responses elicited by metal pollution in the green garden snail, Cantareus apertus: Laboratory and field studies[END_REF] for another snail species, Cantareus apertus. According to the cost of the decontamination [START_REF] Lombi | Remediation of polluted soils[END_REF], the prioritization of the remediation of the soils could be ranked according to the values of the risk index (HQ adj., CR adj. and ERITME) to better assess the adequacy between the uses of the soils and the risks (MEEM, 2017) and to protect terrestrial ecosystems [START_REF] Pauget | Ranking field site management priorities according to their metal transfer to snails[END_REF]. Currently, the management of contaminated soils is predominantly based on HHRA. When HHRA concludes that a risk to humans exists, decontamination or other management methodologies should be used. When remediation solutions are too much expensive or when the economic balance of the site redevelopment project does not cover the management overhead, the project can be abandoned and the site remains unclaimed. The assessment of the environmental risk and the pollutant transfers in the environment developed in this study reveal that, although a risk to humans was identified, the contaminants were not necessarily bioavailable (environmental and/or toxicological bioavailability) to an organism of the soil fauna (see yellows stars in figure 2). Consequently, the anthropic uses of soil and the risks can be assessed by considering the bioaccessibility to humans and the bioavailability to snails (Figure 3). In certain cases, if the IRA shows a higher risk for human health with the current use (ISE > thresholds) but not for the environment (ERITME < threshold), in the future, through developing this methodology to establish a management plan (by using quantitative human health risk assessment), certain sites can be managed by avoiding human activities (e.g., restricting access). In cases such as urban wastelands, the pollutants remain in place to promote land restoration that can increase the biodiversity and/or other alternative uses, such as the production of renewable energy from ecological compensation.

Conclusion

This study provides essential knowledge to begin building bridges between human health and environmental risk indicators in the context of the One Health approach and the increasing global environmental crisis. For the first time, methodologies to determine the level of risk for both risk indicators were applied simultaneously to polluted soils. Strong correlations are found between the values of risk indicators for human and environmental health for the three metal(loid)s studied, but in most cases, their interpretation does not indicate the same level of risk. Unexpectedly, for about two-third of the studied soils both indicators revealed either a confirmed risk or an uncertain level of risk. Another development is that we found that overall polluted soils present greater risk for human than for the environmental bioindicator, with 55 % versus 28 % of the studied soils classified in the proven risk, respectively. The methodology we presented is one way to assess the integrative risk of polluted soils by considering bioaccessibility for humans and bioavailability for bioindicators to better consider the specificity of each site and to protect the health of the ecosystem that encompasses humans and all other life forms.

Declaration of competing interest

The authors declare no conflicts of interest.

Figure 1 :

 1 Figure 1: Relationships between the ratio of [metal(loid)s]soils/SGV(metal(loid)s) (mg kg -1 dw) and the ratio of [metal(loid)s]snails/TGV(metal(loid)s) (mg kg -1 dw) (SGV and TGV were summarized in Louzon et al. (2020b); dotted line represents the identity line y=x).

Figure 2 :

 2 Figure 2: Summary of the HHRA with ISE and ISE adj. and the ERA with ERITME for each of the soils in the study (ISE were adjusted with the oral bioaccessible fraction when HQ or CR were in the area of uncertain risk using the classification in the table 3. Yellow stars indicate soils where human health risk is evidenced or is uncertain and the level of environmental risk is no risked. The result of the risk ranking for ISE in this figure is based on the most negative interpretation of the risk for HQ and CR for all of the metal(loid)s studied. ISE, ISE adj. and ERA with ERITME are presented by colored bar plots in blue, orange and green, respectively).

Figure 3 :

 3 Figure 3: Development of a management plan combining HHRA and ERA (according to results of this study and adapted from the graphical abstract in Louzon et al. (2020a)).

Table 1 : Concentrations of As, Cd and Pb in soils and their bioaccessible fractions (gastric and gastro-intestinal) measured using the UBM test and the concentrations bioaccumulated over 28 days in the viscera of snails (mg kg -1 dw).

 1 Total concentrations of metal(loid)s in soils and their measured bioaccessible fractions are noted as follows for each soil: Csoil(G phase|GI phase).

	Soil	[As]soil	[As]snail	[Cd]soil	[Cd]snail	[Pb]soil	[Pb]snail
	O1 13.4 (38 %|38 %)	0.298	1.32 (95 %|31 %)	5.02	430 (81 %|26 %)	84.6
	O2 11.5 (32 %|29 %)	0.298	1.91 (73 %|21 %)	5.63	320 (68 %|2 %)	43.3
	O3 228 (100 %|49 %)	21.9	257 (100 %|55 %)	333	6256 (100 %|39 %)	856
	O4 74.4 (57 %|38 %)	1.29	48.0 (86 %|25 %)	60.0	5224 (76 %|7 %)	652
	O5 108 (60 %|49 %)	2.25	85.7 (89 %|34 %)	73.6	8971 (100 %|20 %)	731
	O6 139 (80 %|64 %)	19.2	109 (96 %|45 %)	307	2184 (93 %|5 %)	783
	O7 12.8 (35 %|25 %)	0.298	4.20 (92 %|25 %)	18.6	385 (88 %|2 %)	267
	O8 5.64 (21 %|21 %)	0.298	0.64 (85 %|39 %)	5.33	30.7 (66 %|10 %)	5.21
	O9 23.3 (45 %|42 %)	3.93	7.34 (84 %|37 %)	32.0	534 (96 %|2 %)	208
	O10 48.6 (73 %|52 %)	2.31	27.8 (100 %|40 %)	71.9	2233 (100 %|44 %)	503
	O11 27.9 (50 %|49 %)	3.78	18.2 (85 %|36 %)	52.5	1260 (88 %|10 %)	431
	O12 7.74 (14 %|16 %)	0.298	1.42 (73 %|34 %)	12.5	222 (89 %|45 %)	171
	O13 12.6 (27 %|29 %)	0.262	6.50 (74 %|17 %)	28.3	484 (94 %|9 %)	162
	O14 23.4 (38 %|65 %)	2.02	15.4 (87 %|57 %)	46.7	719 (85 %|2 %)	271
	O15 13.5 (38 %|27 %)	0.482	1.30 (78 %|63 %)	6.04	177 (64 %|0 %)	21.0
	O16 5.52 (15 %|16 %)	0.098	0.64 (85 %|32 %)	4.80	17.8 (60 %|0 %)	4.12
	O17 17.8 (11 %|8 %)	0.098	0.200 (77 %|60 %)	2.56	33.7 (41 %|2 %)	1.42
	O18	74.4 (8 %|8 %)	1.39	0.520 (66 %|26 %)	3.20	46.6 (33 %|1 %)	2.56
	O19 10.1 (13 %|14 %)	0.098	4.49 (71 %|28 %)	12.1	117 (72 %|2 %)	37.4
	O20 26.0 (23 %|23 %)	1.98	0.370 (71 %|34 %)	4.59	150 (70 %|2 %)	38.9
	O21 11.3 (49 %|44 %)	2.23	2.32 (83 %|31 %)	11.5	288 (84 %|1 %)	83.7
	O22 3.28 (16 %|18 %)	0.098	0.235 (87 %|57 %)	4.24	19.7 (72 %|18 %)	12.1
	O23 57.0 (30 %|25 %)	1.48	0.102 (82 %|35 %)	2.53	660 (17 %|3 %)	39.1
	O24 12.0 (34 %|24 %)	0.829	3.57 (96 %|29 %)	12.4	303 (86 %|2 %)	35.6
	O26 13.3 (43 %|28 %)	0.098 0.450 (100 %|61 %)	3.20	312 (88 %|3 %)	58.0
	O27 28.5 (24 %|16 %)	0.098	7.43 (73 %|38 %)	8.81	1882 (68 %|7 %)	137
	O28 25.8 (29 %|23 %)	0.098	2.60 (84 %|28 %)	5.52	467 (63 %|9 %)	25.5
	O29 36.1 (24 %|16 %)	0.379	27.2 (49 %|34 %)	28.3	5258 (99 %|32 %)	820
	O30 28.3 (27 %|22 %)	1.16	12.8 (81 %|41 %)	20.4	3241 (97 %|24 %)	730
	Min. 3.28 (8 %|8 %)	0.098	0.102 (49 %|17 %)	2.53	17.8 (17 %|0 %)	1.42
	Med. 23.4 (32 %|25 %)	0.656	3.89 (84 %|34 %)	12.3	426 (84 %|5 %)	110
	Max. 228 (100 %|65 %)	21.9	257 (100 %|63 %)	333	8971 (100 %|45 %)	856

Table 2 : Toxicological reference values (TRV) for As, Cd and Pb (ingestion) in mg kg -1 day -1 (ANSES, 2020; INERIS, 2018) for no carcinogenic risks (with threshold) and carcinogenic risk (CR), toxicity point (TP) (ATSDR, 2015) and ex situ threshold guide values (TGV) for snails in mg kg -1 (Louzon et al., 2020b) Soil TRV with threshold Toxicological target TRV for CR Ex situ TGV TP

 2

	As	0.00045	Skin	1.5	0.364	600
	Cd	0.00036	Kidney	ND	5.60	400
	Pb	0.00063	Kidney	0.0085	9.06	400
	ND: not determined

Table 3 : Interpretation of values of hazard quotient (HQ), cancer risk (CR) (MEEM, 2017) and risk coefficient (RC) (Louzon et al., submitted)

 3

	Risk	HQ	CR	RC
	No	< 0.2 < 10 -6	< 2574
	Uncertain 0.2-5 10 -6 -10 -4 2574-22720
	Proven	> 5	> 10 -4	> 22720

Table 4 : Assessment of human health risk with hazard quotient (HQ), cancer risk (CR) and interpretation the state of the environment

 4 (ISE) (when HQ or CR were in the area of uncertain risk, they were weighted (HQ adj. or CR adj.) by bioaccessible fraction. The result of the ISE is the most conservative level of risk for all the metal(loid)s studied according to the ISE grid of interpretation summarized in table3).

Acknowledgements

The authors would like to thank ADEME for funding the COMBINE study programme (Coupler des indicateurs chiMiques et Biologiques pour une évaluation Intégrative des risques sanitaires et Environnementaux [grant number 1572C0310]) and the ODESSA programme (Optimisation de la gestion Des Sites et Sols pollués par une mesure simple de bioAccessibilité [grant number 1472C0046]). The authors thank Hubert Leprond, Nadia Crini and Francis Douay for their kind advice about our work and Dominique Rieffel and Caroline Amiot for their technical assistance.

no adj. 9.13 x 10 -6 proven proven O12 0.10 5.63 x 10 -6 no. adj. 9.01 x 10 -7 0.02 no TRV no adj. no TRV 1.99 9.10 x 10 -7 3.54 no adj. uncertainty uncertainty O13 0.16 9.18 x 10 -6 no. adj. 2.66 x 10 -6 0.10 no TRV no adj. no TRV 4.34 1.99 x 10 -6 8.16 3.74 x 10 -6 uncertainty proven O14 0.30 1.70 x 10 -5 0.20 1.11 x 10 -5 0.24 no TRV 0.21 no TRV 6.45 2.96 x 10 -6 no adj. 5.03 x 10 -6 proven proven O15 0.17 9.84 x 10 -6 no. adj. The level of risk is interpreted according to three categories: no risk, uncertain risk and proven risk, labelled as green, orange and red, respectively. The level of risk is interpreted according to three categories: no risk, uncertain risk and proven risk, labelled as green, orange and red, respectively.