Farzana Kastury
email: farzana.kastury@mymail.unisa.edu.au

Euan Smith

Emmanuel Doelsch

Enzo Lombi

Martin Donnelley

Patricia L Cmielewski

David W Parsons

Kirk G Scheckel

David Paterson

Martin D De Jonge

Carina Herde

Albert L Juhasz

In vitro, in vivo and spectroscopic assessment of lead exposure reduction via ingestion and inhalation pathways using phosphate and iron amendments

Keywords: Lead, Phosphate amendment, Relative bioavailability, Bioaccessibility, Immobilization, Dust Highlights

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

Introduction

Lead (Pb) exposure has been linked with approximately half a million deaths and nine million disability-adjusted life years worldwide by the Institute for Health Metrics and Evaluation [START_REF] Who | Lead poisoning and health [Online early access[END_REF] .

Cognitive and neurological impairment due to childhood Pb exposure is well documented [2][START_REF] Lanphear | Low-level environmental lead exposure and children's intellectual function: an international pooled analysis[END_REF][START_REF] Surkan | Neuropsychological function in children with blood lead levels < 10 μg/dL[END_REF] and can occur at blood Pb concentration (PbB) as low as 3 µg/dL [START_REF] Chiodo | Neurodevelopmental effects of postnatal lead exposure at very low levels[END_REF] . The two significant pathways for Pb exposure in humans are incidental ingestion of soil/soil borne dust and inhalation of re-suspended indoor surface/house dust [START_REF] Zahran | Linking source and effect: Resuspended soil lead, air lead, and children's blood lead levels in Detroit, Michigan[END_REF] . Solubilization of Pb occurs in the stomach acid (pH 1.5-2.5) [START_REF] Scheckel | Amending soils with phosphate as means to mitigate soil lead hazard: a critical review of the state of the science[END_REF][START_REF] Zhao | Coupling bioavailability and stable isotope ratio to discern dietary and nondietary contribution of metal exposure to residents in mining-impacted areas[END_REF] following soil ingestion or in the lung surfactants and epithelial fluid (pH 7.0) [START_REF] Schultz | Airway surface liquid pH is not acidic in children with cystic fibrosis[END_REF] following dust inhalation. The proposed mechanisms for Pb absorption include passive and facilitated diffusion via the divalent metal transporter in the lungs, duodenum and ileum of the small intestine (pH 7.0) [START_REF] Bannon | Evaluation of small arms range soils for metal contamination and lead bioavailability[END_REF][START_REF] Kanapilly | Measurement of in vitro dissolution of aerosol particles for comparison to in vivo dissolution in the lower respiratory tract after inhalation[END_REF][START_REF] Nemmar | Recent advances in particulate matter and nanoparticle toxicology: a review of the in vivo and in vitro studies[END_REF] . Children are the most at risk of Pb exposure from soil ingestion or dust inhalation because of their higher occurrence of hand-to-mouth activities [START_REF] Scheckel | Amending soils with phosphate as means to mitigate soil lead hazard: a critical review of the state of the science[END_REF] , as well as their increased respiratory frequency, leading to a higher deposition fraction of fine particulate matter (<4 µm) in the respiratory tract [START_REF] Asgharian | Modeling age-related particle deposition in humans[END_REF][START_REF] Rissler | Deposition efficiency of inhaled particles (15-5000 nm) related to breathing pattern and lung function: an experimental study in healthy children and adults[END_REF] .

Reduction in Pb absorption into the systemic circulation using soil amendments (e.g. phosphates, metal oxides, clay minerals) may be a cost effective risk mitigation strategy compared to soil removal, replacement, washing and capping [START_REF] Scheckel | Amending soils with phosphate as means to mitigate soil lead hazard: a critical review of the state of the science[END_REF][START_REF] Hettiarachchi | Soil lead bioavailability and in situ remediation of lead contaminated soils: A review[END_REF] . Particularly, phosphate amendments may reduce Pb bioavailability by promoting the formation of poorly soluble Pbphosphate species [e.g. pyromorphites (Pb5(PO4)3X, where X=OH/Cl/F) and Pb3(PO4)2] 7, [START_REF] Hettiarachchi | Soil lead bioavailability and in situ remediation of lead contaminated soils: A review[END_REF][START_REF] Mele | In situ fixation of metal (loid) s in contaminated soils: a comparison of conventional, opportunistic, and engineered soil amendments[END_REF] . Although Pb immobilization has been demonstrated in humans [START_REF] Maddaloni | Bioavailability of soilborne lead in adults, by stable isotope dilution[END_REF] and animal feeding studies [START_REF] Hettiarachchi | Soil lead bioavailability and in situ remediation of lead contaminated soils: A review[END_REF][START_REF] Maddaloni | Bioavailability of soilborne lead in adults, by stable isotope dilution[END_REF][START_REF] Bradham | Long term in situ reduction in soil lead bioavailability measured in the mouse model[END_REF] , its efficacy is governed by site-specific soil chemistry, Pb speciation and choice of amendment [START_REF] Scheckel | Amending soils with phosphate as means to mitigate soil lead hazard: a critical review of the state of the science[END_REF] .

The predominant focus for Pb immobilization studies thus far has been demonstrating reduction of Pb bioavailability (absorption of Pb into systemic circulation) or bioaccessibility (dissolution of Pb in simulated biological solution) via the ingestion pathway. During the analysis of oral bioavailability/bioaccessibility, <250 µm soil particle fraction is generally used; however, Pb contaminated particulate matter, which may be <10 µm in aerodynamic diameter, may be inhaled in arid mining/smelting impacted regions due to resuspension of dust inside or near households in the vicinity of the mining region [START_REF] Fent | Lead distribution in rats following respiratory exposure to lead-contaminated soils[END_REF][START_REF] Boreland | Lead dust in Broken Hill homes: Effect of remediation on indoor lead levels[END_REF][START_REF] Boreland | Lead dust in Broken Hill homes-a potential hazard for young children?[END_REF] . The concentration of Pb (as well as other toxic elements present) may be several magnitudes higher in the <10 µm particle fraction compared to the < 250 µm counterpart [START_REF] Kastury | An inhalationingestion bioaccessibility assay (IIBA) for the assessment of exposure to metal(loid)s in PM10[END_REF] . Although the mass of inhaled dust may be less than the mass that is incidentally ingested (depending on the local concentration of particulate matter), Fent et al. [START_REF] Fent | Lead distribution in rats following respiratory exposure to lead-contaminated soils[END_REF] reported that bioavailability of Pb from contaminated soil via instillation is significantly higher than ingestion when identical doses for both pathways are used using blood, liver and kidney Pb concentration as bioavailability endpoints. When deposited in the respiratory system, rapid Pb absorption from the lungs of rats instilled with Pb-acetate or Pb contaminated soil may occur [START_REF] Fent | Lead distribution in rats following respiratory exposure to lead-contaminated soils[END_REF] , presumably due to the high gas exchange surface area (70 m 2 ), short distance between the air-blood barrier (0.5 µm) and high vascularization of the respiratory system [START_REF] El-Sherbiny | Inhaled nano-and microparticles for drug delivery[END_REF] . Additionally, particles may reach gastro-intestinal (GI) tract via mucociliary escalator and further dissolve in the stomach, be absorbed in the intestine [START_REF] Kastury | A critical review of approaches and limitations of inhalation bioavailability and bioaccessibility of metal (loid) s from ambient particulate matter or dust[END_REF] , translocate to extra-pulmonary organs (e.g. liver, heart, bone), be retained in the lung for weeks-years 25 and exert additional acute or systemic toxicity [START_REF] Nemmar | Recent advances in particulate matter and nanoparticle toxicology: a review of the in vivo and in vitro studies[END_REF][START_REF] Wallenborn | Systemic translocation of 70 zinc: kinetics following intratracheal instillation in rats[END_REF] . Therefore, incorporating Pb immobilization information for both ingestion and inhalation pathways may be considered a holistic approach when recommending an amendment strategy for risk minimization in arid regions.

Discovered in 1883, Broken Hill city contains the world's largest Pb, zinc (Zn) and silver (Ag) ore deposit known as the Line of Lode [START_REF] Solomon | The Richest Lode: Broken Hill 1983[END_REF] . In addition to the continuous mining since discovery, a brief period of Pb and Zn smelting (1883-1898) [START_REF] Solomon | The Richest Lode: Broken Hill 1983[END_REF] have resulted in widespread Pb contamination in the urban residential area of the Broken Hill city [START_REF] Yang | Bioaccessibility of lead in urban soil of Broken Hill, Australia: a study based on in vitro digestion and the IEUBK model[END_REF] . Dust suppression and Pb exposure mitigation strategies in the Broken Hill residential regions to date include the application of cracker dust in parts of the central southern urban area [START_REF] Yang | Effectiveness of cracker dust as a capping material for Pb-rich soil in the mining town of Broken Hill, Australia[END_REF] and home remediation trial (encapsulation/removal of dust, sealing of cracks in the windows/walls) [START_REF] Boreland | Lead dust in Broken Hill homes: Effect of remediation on indoor lead levels[END_REF] . However, a high risk of chronic childhood Pb exposure was predicted in a recent study by Yang and Cattle [START_REF] Yang | Bioaccessibility of lead in urban soil of Broken Hill, Australia: a study based on in vitro digestion and the IEUBK model[END_REF] .

This study determined the efficacy of Pb immobilization via ingestion and inhalation pathways in Pb contaminated soil from Broken-Hill (Australia) using P and Fe amendments. In addition to assessing Pb exposure reduction using bioaccessibility and bioavailability, changes in Pb speciation in the pre-and post-bioaccessibility residuals and in the lungs were determined using Extended X-ray Absorption Fine Structure (EXAFS) and X-ray Absorption Near Edge Structures (XANES) spectroscopy, while Pb dynamics following in vivo inhalation exposure was assessed via X-Ray Fluorescence (XRF) microscopy.

Materials and methods

Soil collection and physicochemical characterization

Mining/smelting impacted topsoil (0-20 cm) was collected from Broken-Hill, Australia. After drying at 40°C, the soil was sieved to <2 mm and its water holding capacity (WHC), pH [soil(m):water(v)=1:5] and total organic carbon content (LECO TrueMac CNS) was measured (n=3). Subsamples were sieved to <250 µm (incidentally ingestible fraction) and <10 µm (inhalable or dust fraction) (Endecotts Octagon digital shaker). Each fraction (0.1 g, n=3) and a Standard Reference Material (SRM) from the National Institute of Standards and Technology (NIST) 2710a were pre-digested overnight in 5 mL aqua-regia, then digested in a MARS-6 microwave (CEM) using USEPA method 3051 [START_REF]USEPA: Microwave assissted acid digestion of sediments, slidges, soils, and oils[END_REF] . Digested samples were syringe filtered (0.45 µm, cellulose acetate), diluted with MilliQ water and the pseudo-total elemental concentrations analyzed using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) (Agilent 8800) according to USEPA method 6020A [START_REF]EPA: Method 6020A (SW-846): Inductively Coupled Plasma-Mass Spectrometry[END_REF] . The average recovery of Pb from SRM, check values, duplicates and spiked samples were within the limits specified in USPEA method 6020A [START_REF]EPA: Method 6020A (SW-846): Inductively Coupled Plasma-Mass Spectrometry[END_REF] . See Supporting Information (SI) for further details.

Application of amendments

Soil amendment and ageing was conducted according to Juhasz et al. [START_REF] Juhasz | In situ formation of pyromorphite is not required for the reduction of in vivo Pb relative bioavailability in contaminated soils[END_REF] using phosphoric acid (PA, Fisher Scientific), hydroxyapatite (HA, Sigma Aldrich), mono-ammonium phosphate (MAP, Sigma Aldrich), triple super phosphate (TSP, Richgro), bone meal biochar (Charcoal House LLC, Crawford, NE, USA) and synthetic hematite Fe2O3 (Sigma Aldrich). See SI for more details. After the application of amendments (Pb:P/Fe=1:5) at 80% WHC and ageing for two weeks (24±2°C) [START_REF] Juhasz | In situ formation of pyromorphite is not required for the reduction of in vivo Pb relative bioavailability in contaminated soils[END_REF] , soils were dried (40°C) and soil pH determined in a subsample. To remove excess P, soils were leached 5 times with natural rainwater collected from rooftop into rainwater tank (pH 5.8), followed by drying and sieving to recover the <250 µm and <10 µm particle size fractions. Pseudo-total elemental concentrations from each fraction were reanalyzed as described earlier.

Assessment of in vitro bioaccessibility (IVBA)

Oral Pb and Fe IVBA was assessed (n=3) in the <250 µm soil particle size fraction following the Solubility Bioaccessibility Research Consortium (SBRC) assay [START_REF] Kelley | Assessing oral bioavailability of metals in soil[END_REF] . Inhalation Pb and Fe IVBA was assessed (n=3) in the <10 µm soil particle size fraction using the InhalationIngestion Bioaccessibility Assay (IIBA) [START_REF] Kastury | An inhalationingestion bioaccessibility assay (IIBA) for the assessment of exposure to metal(loid)s in PM10[END_REF] using Hatch's solution [START_REF] Berlinger | A study of the bio-accessibility of welding fumes[END_REF] . Percent IVBA was calculated using Eq. 1 given in Kastury et al. [START_REF] Kastury | An inhalationingestion bioaccessibility assay (IIBA) for the assessment of exposure to metal(loid)s in PM10[END_REF] . See SI for more details.

EXAFS assessment of Pb and Fe speciation

Lead speciation was assessed using EXAFS in pre-and post-amendment soil, as well as selected post-IVBA assay residuals (to provide speciation information on the non-bioaccessible fraction). Methodologies described in Kropf et al. [START_REF] Kropf | The new MRCAT (Sector 10) bending magnet beamline at the advanced photon source[END_REF] was used to conduct EXAFS analysisat the Materials Research Collaborative Access Team (MRCAT), Advanced Photon Source of the Argonne National Laboratory, U.S. (beamline 10-ID) . See SI for additional details.

Assessment of oral bioavailability via gavage

In vivo Pb relative bioavailability (RBA) studies were conducted using female Balb/C mice (46 weeks old), according to the Guidelines for the Care and Use of Laboratory Animals [START_REF] Clark | The 1996 guide for the care and use of laboratory animals[END_REF] , with approval from the South Australian Health and Medical Research Institute Animal Ethics Committees (application number SAM268). Based on the Pb IVBA results using SBRC assay, unamended, PA, HA or Fe2O3 amended <250 µm soil particle fraction (20-108 µg of Pb suspended in 180 µL of Pb free water) was administered via gavage to fasted mice (20-25 g) [START_REF] Smith | In vivo-in vitro and XANES spectroscopy assessments of lead bioavailability in contaminated periurban soils[END_REF] .

Blood (~0.5 mL) was collected via cardiac puncture 8 h post exposure, stored at 4°C, and analyzed by ICP-MS after diluting ten-fold in blood diluent solution [START_REF] Technologies | Determination of heavy metals in whole blood by ICP-MS[END_REF] . Equation 1 according to Juhasz et al. [START_REF] Juhasz | In situ formation of pyromorphite is not required for the reduction of in vivo Pb relative bioavailability in contaminated soils[END_REF] was used to calculate Pb RBA(See SI for further details).

Assessment of Pb bioavailability via instillation

Based on the Pb IVBA results of IIBA, PA amended and unamended inhalable dust (< 10 µm fraction) was intra-tracheally instilled using female C57Bl/6 mice (8-10 weeks old) with approval from the Animal Ethics Committee of the Women's and Children's Health Network, Adelaide, South Australia (WCHN AEC project No. AE1044/7/2019). Mice (n=6) were anaesthetized and intubated [START_REF] Cmielewski | Gene therapy for Cystic Fibrosis: Improved delivery techniques and conditioning with lysophosphatidylcholine enhance lentiviral gene transfer in mouse lung airways[END_REF] and a 20 µL bolus dose (0.02 mg of dust/mL suspended in 0.9% NaCl) was administered directly into the trachea over ten seconds via the endotracheal tube.

Mice were exposed for 0.25, 0.5, 1, 2, 4, 8 or 24 h, after which they were humanely killed by CO2 asphyxiation. Blood was collected via cardiac puncture, with Pb concentration analysis undertaken as described above. Lungs and the entire GI tract were removed and immediately frozen at -86°C to prevent change in Pb speciation and stored at -80°C to -20°C until analysis.

Two sets of lungs and GI tracts for each time-point/treatment was reserved for further analysis using XRF described below, while the remaining tissues (n=4) were used to determine Pb concentration. The GI tracts were sectioned into stomachs and intestines. Stomachs were refrozen and freeze dried (Modulyod Freeze Dryer). Lungs, GI tracts and NIST SRM 2976 were digested using nitric acid (70%) in a block digester (A.I. Scientific AIM500) ramping up to a maximum temperature of 180°C [START_REF] Ollson | Influence of co-contaminant exposure on the absorption of arsenic, cadmium and lead[END_REF] . Following digestion and evaporation of acid to ~1-2 mL, samples were diluted with Milli-Q water, syringe filtered (0.45 µm, cellulose acetate) and stored at 4°C until analyzed by ICP-MS. See SI for more details.

XRF and XANES analysis of lungs and GI tracts

In order to retain the morphology of organs during sample preparation for XRF, the mouse organs were freeze dried (Modulyod Freeze Dryer). The GI tracts were thawed, unraveled and positioned onto Kapton tape, while the lungs were dissected and the left lobes were suspended on Lecter coat hangers and frozen prior to freeze drying. The dehydrated organs were sandwiched between ultralene and x-ray fluorescence microscopy (XFM) performed (18.5 keV incident energy, velocity of 2 mm/sec and a pixel size of 20 µm) at the XFM beamline at the Australian Synchrotron. Regions of interest (0.2 x 0.2 mm) from the 24 h unamended and amended lungs were selected for XANES mapping (90 energy steps, mapped at a velocity of 0.5 mm/sec and a pixel size of 2 µm) [START_REF] Etschmann | Speciation mapping of environmental samples using XANES imaging[END_REF] . See SI for additional details.

Statistical analysis

To determine the efficiency of each amendment in reducing ingestion and inhalation exposure, treatment effect ratio (TER) was calculated using Eq. 1 TER = ……….Eq. 1

Significant difference in Pb concentrations in soils post-amendment was determined using One-Way ANOVA (α=0.05). Statistical significances of IVBA and RBA outcomes between unamended and amended soils were assessed using t-test (α=0.05).

Results and Discussion

Elemental concentration and Pb speciation in unamended soil

The pH, TOC and concentrations of elements in the bulk soil (<2 mm), incidentally ingestible (<250 µm) and inhalable (<10 µm) soil particle fractions, are listed in Table 1. The pH of the bulk soil was slightly acidic (5.2±0.08) with low total organic carbon (0.5±0.04%). The concentration of Pb in the <2 mm, <250 µm and <10 µm particle size fractions were 11,656±754 mg/kg, 14,874±157 mg/kg and 62,069±739 mg/kg, exhibiting Pb enrichments of 1.3 and 5.3 fold in the <250 and <10 µm fractions respectively. Among the other notable trace elements of interest, manganese (Mn) and zinc (Zn) were present at an elevated level (3334±754 and 5432±271 mg/kg respectively), while aluminum (Al) and iron (Fe) were the highest among the major elements (19035±960 and 25746±2140 mg/kg). Although metal cations, particularly Mn, Zn, calcium (Ca) are associated with lower PbB level [START_REF] Gulson | Dietary zinc, calcium and nickel are associated with lower childhood blood lead levels[END_REF][START_REF] Kordas | Nutritional status and diet as predictors of children's lead concentrations in blood and urine[END_REF] , combined Pb and Mn exposure has been reported to exacerbate cognitive performance [START_REF] Calderon | Exposure to arsenic and lead and neuropsychological development in Mexican children[END_REF] . Similarly, when inhaled, Zn (18437±174 mg/kg in the <10 µm particle fraction) may potentially cause lung inflammation and fibrosis [START_REF] Adamson | Zinc is the toxic factor in the lung response to an atmospheric particulate sample[END_REF] .

The predominant Pb species in the unamended <250 µm soil particle fraction was anglesite (PbSO4) (66%) and organic matter bound Pb (31%), with the remainder of the Pb was mineral sorbed (e.g. adsorbed onto clays and oxides) (Table 2). Although the proportion of anglesite did not change in the <10 µm soil particle fraction (67%), the majority of the remaining Pb was distributed among clays and oxides (20%), organic matter bound Pb (8%) and plumbojarosites (PbFe 3+ 6(SO4)4(OH)12) (7%).

Effect of soil amendments on pH and Pb/Fe speciation

Phosphoric acid and TSP amendments lowered soil pH from 5.2 to 3.7 and 4.8 respectively, while HA, MAP and Fe2O3 amendments had minimal effect on pH (Table S1). In contrast, biochar amendment resulted in an increase in soil pH to 6.5, presumably as a result of hydroxide ion release from biochar [START_REF] Sneddon | Field trial using bone meal amendments to remediate mine waste derived soil contaminated with zinc, lead and cadmium[END_REF] . Speciation analysis of Pb (EXAFS) in the phosphate amended <250 µm soil particle fractions suggested small increases in anglesites phases with corresponding decreases in organic bound Pb (Table 2). The exception was biochar, where a greater proportion of Pb was organic bound (42%), presumably due to the interaction between Pb and organic ligands in the biochar. In the <10 µm dust fraction, minor redistribution of Pb phases between Pb adsorbed to clays and oxides and organic matter bound Pb were observed after phosphate amendment. Similar to the <250 µm soil particle fraction, biochar amended dust was the exception, where anglesite (45%) and organic bound Pb species (42%) were the dominant Pb phases. Fe speciation in the dust fraction (Table S2) revealed that Fe was present predominantly as ferrihydrite (24-42%) and associated with clay (26-40%), with the remainder distributed as goethite (8-15%), magnetite (5-9%), hematite (5-15%) and maghemite (2-10%).

The formation of Pb-phosphate species (pyromorphite and Pb3(PO4)2) is thermodynamically favored in the presence of H3PO4 0 (at pH of <2.12) and H2PO4 -(at pH between 2.12 to 7.21) [START_REF] Brown | In situ soil treatments to reduce the phyto-and bioavailability of lead, zinc, and cadmium[END_REF] .

Therefore, formation of Pb-phosphate species was expected following PA and TSP amendments (pH 3.7-4.8) (Table S1). However, EXAFS analysis did not detect Pb-phosphates in any phosphate amended soils for either the <250 or <10 µm soil particle size fractions, presumably because the rate of dissolution of anglesite (the dominant phase) is pH independent [START_REF] Zhang | Formation of pyromorphite in anglesite-hydroxyapatite suspensions under varying pH conditions[END_REF] . In the study of anglesite immobilization using of Ca(H2PO4)2.H2O and HA, Cao et al. [START_REF] Carosino | Allergic airway inflammation is differentially exacerbated by daytime and nighttime ultrafine and submicron fine ambient particles: Heme oxygenase-1 as an indicator of PM-mediated allergic inflammation[END_REF] and Zhang and Ryan [START_REF] Zhang | Formation of pyromorphite in anglesite-hydroxyapatite suspensions under varying pH conditions[END_REF] reported a rapid dissolution of anglesite in aqueous systems, with the detection of chloropyromorphite. However, more complex interactions may occur in natural soil system, limiting Pb immobilization in situ (e.g. transient increases in soluble Pb, incomplete formation or dissolution of Pb-phosphates due to inadequate Pb dissolution, competition from high cation concentrations with phosphate for sorption sites) [START_REF] Butkus | Reevaluation of Phosphate as a Means of Retarding Lead Transport from Sandy Firing Ranges[END_REF][START_REF] Debela | Organic acid-induced release of lead from pyromorphite and its relevance to reclamation of Pbcontaminated soils[END_REF][START_REF] Karna | Point of Zero Charge: Role in Pyromorphite Formation and Bioaccessibility of Lead and Arsenic in PhosphateAmended Soils[END_REF] . Higher phosphate application rates may be necessary in future studies in Broken Hill soil to compensate for competition posed by cations (e.g. Al, Ca, Fe, Mn, Zn).

Treatment efficacy -ingestion pathway Pb IVBA and residual Pb speciation following gastric phase (SBRC-G) assessment

Pb IVBA in unamended soil was 88.6±0.8% when assessed in the SBRC-G phase (Figure 1A).

This value was at the high end of the range (23.7-89.3%) previously reported in Yang and

Cattle [START_REF] Yang | Bioaccessibility of lead in urban soil of Broken Hill, Australia: a study based on in vitro digestion and the IEUBK model[END_REF] for Broken-Hill topsoil, who reported an average gastric phase Pb IVBA value of 61.2±14%. The lower average value in Yang and Cattle [START_REF] Yang | Bioaccessibility of lead in urban soil of Broken Hill, Australia: a study based on in vitro digestion and the IEUBK model[END_REF] most likely reflected the assessment of soils from a large geographic area with diverse Pb speciation influencing IVBA outcomes.

In contrast, soil was collected near a former Pb ore crushing area in this study and contained anglesite (PbSO4) as the dominant phase, which is a common occurrence in mining impacted soils with acidic pH and results from the oxidation of galena 7 . Anglesite's solubility over a wide range of pH (2-7) [START_REF] Zhang | Formation of pyromorphite in anglesite-hydroxyapatite suspensions under varying pH conditions[END_REF] may have resulted in higher Pb IVBA results.

No significant reduction (P>0.05; Figure 1A) in Pb IVBA was observed in P and Fe amended soil (TER=1.02-1.08; Table S3) using SBRC-G phase, which is similar to the results reported for amended mining soil when assessed at pH 1.5 [START_REF] Mele | In situ fixation of metal (loid) s in contaminated soils: a comparison of conventional, opportunistic, and engineered soil amendments[END_REF][START_REF] Juhasz | Predictive capabilities of in vitro assays for estimating Pb relative bioavailability in phosphate amended soils[END_REF][START_REF] Scheckel | Determining speciation of Pb in phosphate-amended soils: Method limitations[END_REF] . Pb speciation in SBRC-G residual soil identified that the majority of anglesite was solubilized (Figure 1B), which was expected due to anglesite's propensity for solubilization under low pH conditions [START_REF] Zhang | Formation of pyromorphite in anglesite-hydroxyapatite suspensions under varying pH conditions[END_REF] .

Pb IVBA and residual Pb speciation following intestinal phase (SBRC-I) assessment

When IVBA assays were modified to reflect intestinal phase conditions (i.e. addition of bile and pancreatin, pH adjustment to 7.0), Pb IVBA in the unamended soil was reduced to 16.6±1.3%. The decrease in Pb IVBA corresponded to a 88% decrease in Fe IVBA (Figure S2A), resulting from the precipitation of amorphous Fe species following oversaturation of Fe during the pH change [START_REF] Mercer | Removal of arsenic from high ionic strength solutions: effects of ionic strength, pH, and preformed versus in situ formed HFO[END_REF] . During the transition from the gastric to intestinal phases, the increase in pH may have caused Pb to precipitate and become re-adsorbed to the Fe oxides and/or soil matrix [START_REF] Smith | In vivo-in vitro and XANES spectroscopy assessments of lead bioavailability in contaminated periurban soils[END_REF] . A corresponding increase in mineral sorbed Pb species in post-extraction intestinal phase residuals (Figure 1B) supports the hypothesis that Pb co-precipitates with Fe, as suggested by Smith et al. [START_REF] Smith | In vivo-in vitro and XANES spectroscopy assessments of lead bioavailability in contaminated periurban soils[END_REF] . A significant reduction (p<0.001) in Pb IVBA was observed for all P amended soils (TER=0.02-0.1). However, Pb IVBA in Fe2O3 amended soil was similar to the unamended soil (18.0±0.7%), suggesting that Pb immobilization did not occur in vitro.

Among the phosphate amended soils, the lowest TER following SBRC-I assessment was obtained for HA and biochar amended soil (0.02 and 0.04 respectively), followed by PA, MAP and TSP (TERs of 0.1). As HA and biochar exhibit low solubility in water, higher Pb:P ratios

(1:5.3 and 1:4.1 respectively) were observed in the <250 µm amended soil factions following rainwater leaching used to remove the excess P after the two-week incubation period (Table S1). Consequently, due to the dissolution of P during SBRC-G assessment, higher concentrations of P were available to react with Pb in the SBRC-I phase, resulting in lower TER. This is also supported in Figure 1B, which shows a 2.2-2.3 fold increase in Pb-phosphate species in the post-IVBA residuals of SBRC-I in all phosphate treated soils but not in the soil amended with Fe2O3. Although only PA amended post-IVBA residual was tested to represent water soluble amendments, presumably Pb-P would form in the MAP and TSP amended residuals as well.

Pb RBA

Although assessment of Pb IVBA using SBRC-I determined that all phosphate amendments were effective in reducing Pb IVBA due to the presence of Pb-phosphates in the nonbioaccessible residuals, Scheckel et al. [START_REF] Scheckel | Amending soils with phosphate as means to mitigate soil lead hazard: a critical review of the state of the science[END_REF] cautioned the use of IVBA outcomes (extracted at pH > 1.5) in interpreting phosphate amendment efficacies due to the potential formation of pyromorphite in vitro as a result of the pH change. Therefore, in addition to IVBA, the efficacy of Pb immobilization was also assessed using Pb RBA in unamended and selected amended soils (PA, HA, Fe2O3). In the unamended soil, Pb RBA was 47.9±2.6%, which was higher compared to similar studies utilizing mining/smelting impacted Australian soils [START_REF] Juhasz | In situ formation of pyromorphite is not required for the reduction of in vivo Pb relative bioavailability in contaminated soils[END_REF][START_REF] Smith | In vivo-in vitro and XANES spectroscopy assessments of lead bioavailability in contaminated periurban soils[END_REF] . Low Pb RBA may occur when sparingly soluble Pb species are present, such as galena or Pbphosphates [START_REF] Davis | Factors controlling lead bioavailability in the Butte mining district, Montana, USA[END_REF][START_REF] Ruby | Advances in evaluating the oral bioavailability of inorganics in soil for use in human health risk assessment[END_REF] . However, the dominant Pb phase, anglesite, may have contributed to the high Pb RBA observed in this study because of its solubility in a wide pH range (2-7) [START_REF] Zhang | Formation of pyromorphite in anglesite-hydroxyapatite suspensions under varying pH conditions[END_REF] .

When soil was amended with PA and HA, Pb RBA was reduced to 21.1±6.2 % (TER=0.79) and 35.8±10.7% (TER=0.74) respectively, which supports the hypothesis of Juhasz et al. [START_REF] Juhasz | In situ formation of pyromorphite is not required for the reduction of in vivo Pb relative bioavailability in contaminated soils[END_REF] that Pb immobilization may occur in vivo, even when in situ Pb-phosphate formation did not occur.

Bradman et al. [START_REF] Bradham | Long term in situ reduction in soil lead bioavailability measured in the mouse model[END_REF] also reported that when Pb speciation in ingested soil was compared to feces following administration in mice, Pb speciation changed during the transit through the GI tract.

Despite both phosphate amendments reducing Pb RBA, a significant reduction was only observed in the PA amended soil (p<0.01). High variability in Pb RBA was observed in the HA amended soil, which may be attributed to the slow dissolution kinetics of HA in the stomach, resulting in the formation of a layer of Pb phosphate species on the outside of the HA particles, reducing the surface area available for reactions with Pb [START_REF] Zhang | Formation of pyromorphite in anglesite-hydroxyapatite suspensions under varying pH conditions[END_REF] . In contrast to the P amended sample, there was no reduction in Pb RBA in the Fe2O3 amended sample (TER=1.2).

Treatment efficacy -inhalation pathway Pb IVBA and residual Pb speciation following IIBA lung phase (IIBA-L) assessment

In the unamended dust fraction, Pb IVBA was 61.7±0.5% during the IIBA-L phase (Figure 2A). High Pb solubility in Hatch's solution was expected, given that anglesites was the dominant Pb phase with solubility in a wide pH range [START_REF] Zhang | Formation of pyromorphite in anglesite-hydroxyapatite suspensions under varying pH conditions[END_REF] and the high concentration of ligands and surfactants present in the extracting solution. The major speciation change in the IIBA-L phase residual was the reduction in anglesite concentration, indication mobilization of anglesites in Hatch's solution (Figure 2B). Lung phase Pb IVBA in unamended dust was lower compared to the results obtained for the SBRC-G assessment (<250 µm soil particle fraction) reported in the preceding section, which may be attributed to the lower pH of the latter.

However, despite IIBA-L and SBRC-I phases were conducted at neutral pH, IIBA-L Pb IVBA was 3.7 fold higher in the former. Lung surfactants and chelators in Hatch's solution may have rapidly transported the solubilized ions away from the surface of the dust particles, preventing their precipitation during IIBA-L assessment [START_REF] Kastury | An inhalationingestion bioaccessibility assay (IIBA) for the assessment of exposure to metal(loid)s in PM10[END_REF] . Additionally, the use of longer extraction period in IIBA-L (24 h) compared to SBRC-I (4 h) allowed a greater dissolution of Pb from the matrix, which was also observed in Kastury et al. [START_REF] Kastury | An inhalationingestion bioaccessibility assay (IIBA) for the assessment of exposure to metal(loid)s in PM10[END_REF] when assessing other mining/smelting impacted dusts.

In PA, MAP and TSP amended dust, Pb IVBA was significantly reduced from 61.7 ± 0.5% in the unamended dust to 20.0±0.9% (PA) -29.5±1.1% (TSP) following assessment using IIBAL (p<0.01, TER=0.33-0.48). In contrast, no significant reduction in Pb IVBA was observed in HA, bone meal biochar and Fe2O3 amended dusts (TER=0.9-1.1), presumably due to the reduced anglesite dissolution observed in IIBA-L residuals (Figure 2B). The presence of Pbphosphates in the MAP and TSP amended IIBA-L residuals suggested the possibility that an outer layer of Pb-phosphates may have formed surrounding Pb particles during the assay, reducing further Pb dissolution 58 .

Pb IVBA and residual Pb speciation following IIBA lung + intestinal phase (IIBA-I) assessment

Following a 24 h extraction in lung fluid, dust particles were transitioned to gastric (pH 1.5, 1 h) and intestinal solutions (pH 7.0, 4 h) to simulate the clearance of particles from the lungs and their passage through the GI tract. In the unamended dust, Pb IVBA in IIBA-I was 74.5±0.8% (Figure 2A). Solubilization of Pb in the acidic pH of the preceding gastric phase may be attributed to the higher Pb IVBA following IIBA-I extraction compared to IIBA-L, which was also evident in the reduction in residual anglesite and an increase in residual plumbojarosites (Figure 2B). Similar to the SBRC-I phase, co-precipitation with Fe during the transition from IIBA-G to IIBA-I may be attributed to the increase in residual plumbojarosites [START_REF] Smith | In vivo-in vitro and XANES spectroscopy assessments of lead bioavailability in contaminated periurban soils[END_REF] .

An important observation during the IIBA-L phase was that Pb IVBA was reduced from 74.5±0.8% in the unamended dust to 44.9±2.1% -59.3±1.8% in PA, MAP and TSP amended dust (p<0.01, TER: 0.67-0.80), while no reduction was observed in HA, biochar and Fe2O3 amended dust (TER 0.97-1.13). EXAFS analysis identified Pb-phosphate species in the IIBAI residuals of PA, MAP and TSP amended dust, which explains the reduction in Pb IVBA in the IIBA-I phase. The combined results of Pb IVBA reduction in phosphate amended dust using IIBA-L and IIBA-I phases indicated that water soluble phosphate amendments may be the most suitable for amending Pb contaminated soil. This result is particularly germane in arid mining/smelting impacted regions of Australia, where exposure via aeolian transport of dust may be considered a significant contributor to childhood PbB [START_REF] Fent | Lead distribution in rats following respiratory exposure to lead-contaminated soils[END_REF][START_REF] Boreland | Lead dust in Broken Hill homes: Effect of remediation on indoor lead levels[END_REF][START_REF] Boreland | Lead dust in Broken Hill homes-a potential hazard for young children?[END_REF] .

Pb bioavailability following dust instillation into mouse lungs

Based on the Pb IVBA results, unamended and PA amended dust was instilled into mice lungs to ascertain whether a reduction in Pb absorption occurs in vivo. Reduction of inhalation bioavailability using phosphate amendment was deemed an important part of overall Pb exposure reduction in Broken Hill because human studies have demonstrated that up to 95% of the Pb in fine particles (<0.1 µm) may be absorbed upon inhalation [START_REF] Hursh | Fate of 212 Pb inhaled by human subjects[END_REF][START_REF] Wells | Deposition in the lung and uptake to blood of motor exhaust labelled with 203Pb[END_REF] . A study conducted by Borelnad et al. [START_REF] Boreland | Lead dust in Broken Hill homes-a potential hazard for young children?[END_REF] reported an influx of 166-1104 µg of P /m 2 during a 30 day period, which indicated that windblown dust in an arid environment such as Broken Hill may potentially contribute to childhood PbB via inhalation. The choice of instillation to assess Pb exposure as a surrogate to inhalation assays enabled a more precise delivery of small sample masses [START_REF] Fent | Lead distribution in rats following respiratory exposure to lead-contaminated soils[END_REF] compared to insufflation of dry dust, allowing direct comparison of Pb absorption in unamended and PA amended dust. Following exposure to the unamended dust, PbB increased rapidly from 0.29 µg/L (time=0) to 18.2±4.4 at 0.25 h and 49.7±5.1 µg/L after 2 h (Figure 2C).

Subsequently, a slower rate of Pb absorption between 2-8 h resulted in a peak PbB of 80.1±11.2 µg/L at 8 h. At the end of 24 h, a moderate decrease in PbB was observed (71.5±9.8 µg/L). The rapid absorption of Pb from Pb contaminated dust in this study agrees with the results reported in Fent et al. [START_REF] Fent | Lead distribution in rats following respiratory exposure to lead-contaminated soils[END_REF] , who instilled dust from shooting range soil into rat lungs and reported a high Pb absorption, which peaked at 24 hours and slowly declined until 96 hours without PbB returning to baseline. Although the maximum exposure time-frame of 24 hours in this experiment was not sufficiently long for PbB to return to baseline, this information, together with the bio-distribution data obtained via the XRF images, yielded valuable insight into the bioavailability of Pb following exposure to Pb contaminated dust. Tricolor XRF images of lungs instilled with the unamended sample showed that Pb (red) became distributed throughout the lungs between 0.5 -2 h (Figure 3A), presumably aided by gravity and lung surfactants [START_REF] Brain | Pulmonary distribution of particles given by intratracheal instillation or by aerosol inhalation[END_REF][START_REF] Geiser | Deposition and biokinetics of inhaled nanoparticles[END_REF] .

While Fe (green) was mostly observed as associated with Pb (indicated by yellow), Ca (blue) was mostly observed in the upper respiratory tract, incorporated in the tracheal rings.Metal sulphates (e.g. anglesite) have been suggested to be water-soluble, therefore potentially bioavailable in the lungs [START_REF] Kyotani | Determination of water and acid soluble components in atmospheric dust by inductively coupled plasma atomic emission spectrometry, ion chromatography and ion-selective electrode method[END_REF][START_REF] Wallenborn | Systemic translocation of particulate matter-associated metals following a single intratracheal instillation in rats[END_REF] . As anglesite was the predominant Pb phase in the unamended dust, anglesite dissolution may have resulted in the majority of Pb absorption via the air-blood barrier.

Tricolor images of the GI tracts showed that Pb appeared in the stomach 8 h post instillation with the unamended dust (Figure 4A), presumably due to particle clearance from the lungs by the mucociliary escalator and the diffusion of Pb throughout the stomach. This was confirmed by Pb concentrations in the lungs (PbL) and stomach (PbS) in Figure 4C, where PbL decreased from 1.1±0.2 µg/organ at 0.25 h to 0.5±0.1 µg/organ at 8 h, while PbS increased from 0.06±0.1

µg/organ at 0.25 h to 1.5±0.3 µg/organ at 8 h.Low concentrations of Pb was detected in this compartment at 24 h (Figure 4A), presumably due to precipitation of non-absorbed Pb.

Detection of Pb in the small intestinal tracts suggested that in addition to Pb absorption in the lungs, it is possible that a fraction of the PbB during 8-24 h resulted from the solubilization of Pb in the stomach, followed by absorption in the small intestine [START_REF] Bannon | Evaluation of small arms range soils for metal contamination and lead bioavailability[END_REF] .

In contrast to the unamended dust, PbB in mice instilled with PA amended dust showed a slower rate of Pb absorption; PbB at 0.25 h being 3.7±0.5 µg/L and reaching a maximum of 48.7±3.7

µg/L at 24 h (Figure 2C). A comparison of the AUC of the unamended and PA amended dust suggested a 55.8±1.6% decrease in the total Pb absorbed into the systemic circulation as a consequence of PA amendment over 24 h. Although it was not clear from Figure 3C whether PbB had peaked during 8-24 h, or if it would continue to increase beyond 24 h, the reduction in vivo lies within the 32.4-67.5% reduction interval in the Pb IVBA using IIBA-L and IIBA-I phases. Additionally, because the reduced Pb absorption was more prominent during the first 8 h of the assay, it was theorized that similar to the Pb-phosphates formation in the small intestine during the gavage study described above, Pb-phosphates may have formed in the lung owing to its neutral pH. Further experiment with longer time frame may be conducted to estimate AUC once PbB has returned to baseline with dose normalization using Pb acetate as a reference dose.

XRF images of lungs and GI tracts depict Pb dynamics in the PA amended sample (Figures 3B and4B). In contrast to the unamended dust, Pb particles from PA amended dust was dispersed more rapidly at 0.25 h. The discrepancy between the dispersion rates most likely resulted from inter-animal variation, which is inherent in instillation experiments [START_REF] Kreyling | Air-blood barrier translocation of tracheally instilled gold nanoparticles inversely depends on particle size[END_REF] . The quicker dispersion of PA amended dust in the lungs may have contributed to its earlier detection in the stomach (4 h; Figure 4B). PbS increased from 0.41±0.1 µg/organ at 0.25 h to 5.8±1.6 µg/organ at 4 h and peaked at 11.1±2.1 µg/organ after 8 h (Figure 4D), supporting the aforementioned suggestion that Pb from PA amended dust demonstrated reduced absorption in the lungs.

To further test this theory, a 0.2 x 0.2 mm region of the lung receiving the unamended and PA amended dust at 24 h was analyzed using XANES to investigate Pb speciation in the residual Pb particles. Within these regions, spectra corresponding to high and low Pb concentrations were obtained (unamendedhigh, unamendedlow, PA amendedhigh and PA amendedlow, Figure S4).

The spectra and their corresponding LCF fits are depicted in Figure 3C

Figure 1 :

 1 Figure 1: (A) Pb IVBA (%) using the Solubility Bioaccessibility Research Consortium (SBRC) assay where SBRC-G = extraction using the gastric phase and SBRC-I = extraction using the gastro-intestinal phase. (B) Pb speciation of pre-and post-SRBC assay residuals. (C) Pb relative bioavailability (RBA) in unamended and selected amended soil. PA = phosphoric acid, HA = hydroxyapatite, MAP = monoammonium phosphate, TSP = triple super phosphate, numbers on the top of bars of (A) and (C) represent treatment effect ratio (TER), asterix represents significance differences between unamended and amended soil ** = p < 0.01 and *** = p <0.001 and n.d. = not determined.

Figure 2 :

 2 Figure 2: (A) Pb IVBA (%) using the Inhalation-Ingestion Bioaccessibility Assay (IIBA) where IIBA-L = extraction using the lung phase, IIBA-I = extraction using the lung+GI phase. (B) Pb speciation in pre-and post-IIBA assay residuals. (C) Blood Pb concentration following instillation of unamended and PA amended soil. PA = phosphoric acid, HA = hydroxyapatite, MAP = monoammonium phosphate, TSP = triple super phosphate, numbers on the top of bars of (A) represent treatment effect ratio (TER), asterix represents significance differences between unamended and amended soil * = p < 0.05, ** = p < 0.01 and *** = p <0.001 and n.d. = not determined.

Figure 3 :

 3 Figure 3: (A) and (B) 2D Elemental map (Pb = red, Fe = green and Ca = blue) using X-ray

Figure 4 :

 4 Figure 4: (A) and (B) 2D Elemental mapping (Pb = red, Fe = green and Ca = blue) in the

Table 1 :

 1 , while Pb speciation (weighted %) is provided in Figure3D. Residual Pb speciation in the unamendedhigh was dominated by anglesites (61.3%) with the remainder consisting of Pb phosphates (38.8%), while that in the PA amendedhigh was distributed as anglesite (48.5%) and Pb phosphates (51.5%), a 1.3 fold increase in Pb-phosphates in the PA amended dust residuals. . Similarly, when residual Pb speciation in the unamendedlow and PA amendedlow were assessed, a 1.3 fold increase in Pb-phosphate was detected in the lungs receiving the PA amended dust ( 39% in the unamended and 50.6% in the PA amended). The remainder of the unamendedlow Pb was comprised of 51% anglesite and 10% mineral sorbed, while that in PA amendedlow was 27.3% anglesite and 22.2% mineral sorbed. It is likely that similar to the results observed in the IIBAL residuals, the Pb ions on the surface of anglesites reacted to P and Cl in the lung lining fluid forming a coating of chloropyromorphite, preventing subsequent dissolution, which was also reported in Wragg and Klinck during an IVBA study using SLF 58 . It is noteworthy that although a small region of the lung was analyzed for residual Pb speciation, the formation of Pbphosphates was 1.7 fold higher in the PA amended dust. Therefore, it is possible that even though Pb-phosphates did not form in situ, the presence of soluble P in the dust at the time of to have limited impact on lowering children's PbB when initial Pb levels were not high[START_REF] Boreland | Lead dust in Broken Hill homes: Effect of remediation on indoor lead levels[END_REF] . Therefore, in urban soils and in arid regions, Pb immobilization using water-soluble phosphate sources may be an effective remediation strategy to mitigate Pb exposure. Physico-chemical characterisation of the soil particle sizes fractions and amendments. N.D. = not determined

	instillation increased the likelihood of Pb-phosphate formation thereby reducing Pb absorption
	in the lungs.
	To the best of our knowledge, this is the first study to report a reduction of Pb exposure using
	phosphate amended dust via instillation, although a longer study may be required for PbB to
	return to (near) baseline. Nevertheless, this study highlighted that although P amendments with
	varying solubility reduced Pb exposure via the oral pathway, water soluble amendments were
	more effective in immobilizing Pb via the inhalation pathway, with important implications of
	improving Pb exposure mitigation strategies worldwide. As particle retention in the lung is
	longer than that in the GI tract, future inhalation bioavailability studies should focused on

longer term bio-assays utilizing dust with different Pb phases to ascertain the influence of Pb speciation on Pb absorption in the lungs and GI tracts. Although the Pb concentration in urban soil typically <1000 mg/kg, urban soil Pb has been linked with children's PbB 66,67 .

Additionally, remediation of homes to seal houses to prevent Pb entry via fugitive dust has been shown

Table 2 :

 2 Pb speciation (weighted %) in the <250 and <10 µm soil particle fraction. PA = phosphoric acid, HA = hydroxyapatite, MAP = monoammonium phosphate, TSP = triple super phosphate.

	Adsorbed	Organic	Anglesite	Plumbojarosite	Pbphosphates	Galena	R
	into clays and	matter bound	(PbSO4)	(PbFe6(SO4)4(OH)12)	(pyromorphite	(PbS)	factor
	oxides				+ Pb3(PO4)2)
			< 250 µm soil particle fraction

Acknowledgements

The authors gratefully acknowledge the help of Susie Ritch, Ranju Karna and Thea Read during the data analysis. Although EPA contributed to this article, the research presented was not performed by or funded by EPA and was not subject to EPA's quality system requirements.

Consequently, the views, interpretations, and conclusions expressed in this article are solely those of the authors and do not necessarily reflect or represent EPA's views or policies. MRCAT operations are supported by the Department of Energy and the MRCAT member institutions.

Funding

Farzana Kastury acknowledges the Commonwealth Government of Australia, Research Training program scholarship (RTPd), University of South Australia for the VC and President's Scholarship and the MF & MH Joyner Scholarship in Science. Funding for aspects of the project was provided by the Women's and Children's Hospital Foundation. E. Doelsch has received funding from European Unions' Horizon 2020 research and innovation program under the Marie Skodowska-Curie agreement N795614. X-ray fluorescence microscopy research (proposal AS181/XFM/13096) was undertaken on the X-ray Fluorescence Microscopy beamline at the Australian Synchrotron, part of ANSTO. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357.

 S1: Change in pH, Pb and P concentration before and after soil amendment, Table S2: Fe speciation (Weighted %) in the < 10 µm particle size fraction, Table S3: Summary of treatment effect ratios (TER) for Pb amended soils.


Gabriel Jarry
email: jarry.gabriel@enac.fr

Daniel Delahaye
email: daniel.delahaye@enac.fr

Eric Feron
email: feron@gatech.edu

Trajectory APproach AnalysiS: A Post-operational Aircraft Approach Analysis Tool

Keywords: Flight path safety management, safety events, atypical approaches, anomaly detection, flight data monitoring

In aviation, safety has always been a key issue to reduce the number of incidents and accidents. Nowadays, it is even more important since the air traffic increases every year, and is predicted to reach 7.8 billion passengers worldwide in 2036. Flight safety offices aim at enhancing safety, analyzing past events, and preventing potential critical occurrences. This paper presents TAPAS, a post-operational aircraft approach analysis tool. The software offers an interactive analysis of aircraft approach energy management. The software uses and validates an existing atypical approach detection algorithm on flight data record and flight safety office events from airlines. Various correlations and analysis are conducted to illustrate the potential benefits of this methodology.

I. INTRODUCTION

Approach and landing accidents (i.e. accidents that occur during the initial approach, the intermediate approach, and during landing) represent every year 47% of the total accidents and 40% of fatalities [START_REF] Jackman | Nearly Half of Commercial Jet Accidents Occur During Final Approach, Landing[END_REF]. Moreover, a great majority of accidents features unexpected events that makes the trajectory differ significantly from nominal approaches such as atypical speed or atypical altitude [START_REF] Tremaud | Getting To Grips With ALAR[END_REF]. In addition, airports Terminal Maneuvering Areas (TMA) and Control Traffic Regions (CTR) are characterized by a dense air traffic flow with high complexity. This complexity will surely increase in the future since IATA forecasts growth of air passenger worldwide from around 4 billion today, up to 7.8 billion in 2036 [START_REF] Iata | 2036 Forecast Reveals Air Passengers Will Nearly Double to 7.8 Billion[END_REF]. Consequently, there is a crucial need for aircraft atypical approach detection method and analysis tools.

The French Civil Aviation Authority has launched in 2006 a national safety program in order to satisfy the International Civil Aviation Organization (ICAO) safety requirement. These safety states program distinguishes undesirable events in different categories such as Non-Stabilized Approaches (NSA), from ultimate events such as Control Flight Into Terrain (CFIT) or mid-air collisions. Undesirable events may lead to final events and therefore jeopardize the safety or reduce airfield capacity. Their identification and detection is an important issue.

Airline flight safety offices aim at analyzing operated flight in order to enhance safety. Usually, each flight is analyzed and monitored if it overpasses safety limits defined by the airlines. The flight analysis enables modifying their own standards operation procedures and the inherent pilot behaviors when it brings safety issues. In general, airlines use dedicated flight data monitoring software, which enables individual or general statistics and analysis.

In this paper, a post-operational tool called Trajectory APproach Analysis (TAPAS) dedicated to the analysis of aircraft energy management during approach and landing is presented. This software is developed with French airlines in order to propose an alternative detection of atypical approaches and atypical energy management during landing. This detection is based on a mathematical atypical trajectory scoring [START_REF] Jarry | Aircraft atypical approach detection using functional principal component analysis[END_REF]. This paper aims at validating the atypical trajectory detection methodology with airline on-board data records and airline flight office analysis. In addition, this study investigates to what extent this methodology could be beneficial to enhance safety since it gives an alternative analysis of safety during approaches.

The paper is divided into four sections. Section II survey the existing litterature. Section III explains the methodology and the data used in the software. Section IV describes TAPAS software organisation and interactions. Finally, Section V illustrates results and analysis produced by the software.

II. STATE OF THE ART

An exhaustive state of the art on aircraft atypical approach detection is presented in [START_REF] Jarry | Aircraft atypical approach detection using functional principal component analysis[END_REF], only major and most relevant publications are surveyed here. Detecting an atypical behavior is a well-known problem referred as to anomaly detection. Anomaly detection has been investigated for a long time: it consists in finding samples from a data set that do not comply with the overall behavior. Among the various methods available, the Multiple Kernel Anomaly Detection (MKAD) technique [START_REF] Das | Multiple kernel learning for heterogeneous anomaly detection: algorithm and aviation safety case study[END_REF] is one of the most efficient algorithms. It was developed to detect anomalies in heterogeneous data (i.e involving both discrete and continuous data), and has been used to detect anomalies in aircraft approach parameters from on-board data. Another kernel-based approach to study onboard aircraft parameters is detailed in [START_REF] Chrysanthos | Kernel methods for flight data monitoring[END_REF]. Neural network auto-encoder reconstruction error can also be used to detect abnormal behavior [START_REF] Sakurada | Anomaly detection using autoencoders with nonlinear dimensionality reduction[END_REF], [START_REF] Zhou | Anomaly detection with robust deep autoencoders[END_REF]. Other anomaly detection techniques using information geometry and functional representation have also been investigated. In her thesis [START_REF] Barreyre | Statistiques en grande dimension pour la détection d'anomalies dans les données fonctionnelles issues des satellites[END_REF], Barreyre introduces different outlier detection methods in space telemetries. More recent techniques use Generative Adversarial networks to detect atypical trajectories [START_REF] Jarry | On the use of generative adversarial networks for aircraft trajectory generation and atypical approach detection[END_REF].

III. METHODOLOGY AND DATA A. Methodology

In this paper, the atypical trajectories are detected using a process that combines a Functional Principal Component Analysis (FPCA) and an outlier scoring on a sliding window [START_REF] Jarry | Aircraft atypical approach detection using functional principal component analysis[END_REF]. The main idea is to focus on the aircraft total energy management, or on an approximation of the total energy while studying radar data. Indeed, the major issue an aircraft is facing in order to land is managing an excess of energy. Excess of energy corresponds to situations where an aircraft is, for example, too high on glide, resulting in too high potential energy, or with a high speed owing to tailwind in final approach, or late power reduction resulting in high kinetic energy. High energy implies, first to detect the excess of energy, and second to manage the energy so as to recover a nominal state.

FPCA is an information geometry tool that extends the well known Multivariate Principal Component Analysis (MPCA) [START_REF] Jolliffe | Principal component analysis[END_REF] to the functional setting. It was extended by Deville [START_REF] Deville | Méthodes Statistiques et Numériques de l'analyse Harmonique[END_REF] and Dauxois [START_REF] Dauxois | Les Analyses Factorielles En Calcul Des Probabiblités et En Statistique: Essai d'étude Synthétique[END_REF], [START_REF] Dauxois | Asymptotic Theory for the Principal Component Analysis of a Vector Random Function: Some Applications to Statistical Inference[END_REF]. When data are functions sampled from an underlying stochastic process, FPCA enables dimensionality reduction by estimating the Karhunen-Loève decomposition. With this decomposition, the trajectories can be represented by their decomposition coefficients on the principal component basis, and considered as a small dimension vector. This process is described as.

Γ(t) = γ(t) + +∞ j=1 b j φ j (t) (1)
Where each curve Γ is considered as the weighted sum of a mean curve γ plus the principal components φ j by defining the orthogonal basis that maximizes the explained variance in the first dimensions. Usually, the decomposition is truncated to keep an amount of variance, which also implies further dimensionality reduction.

The process, illustrated in Figure 1 is applied to each window slides to enable a local anomaly scoring. It is divided into three steps. First, the FPCA decomposition is applied to a window slide of total energy curves set. Second, an outlier score is computed for each sample. Third, the outlier scores are attributed to the corresponding trajectories.

Finally, trajectories with an atypical score above some threshold value (0.6 in the tests) during more than some reference duration (2NM in the tests) are considered to be atypical

B. Data

The data used in this paper are composed with flight data record and flight data events from airline flight data recorder and safety office analysis for flights between March 2017 and June 2019. Each aircraft records on-board parameters; this data gives a complete description of the aircraft configuration and flight situation at every moment. The data were extracted with a frame rate of 4 seconds to fit with the radar recording rate used in the algorithm [START_REF] Jarry | Aircraft atypical approach detection using functional principal component analysis[END_REF]. These data are further analyzed by the safety office, and safety events are recorded when a parameter exceeds some predefined limit. Safety events are separated per flight phases, and intensity levels (high, medium, and low). The airport runways and QFUs studied in the analysis are detailed in Table I IV. TAPAS A. Software Organization TAPAS software is divided into four sections. The first section called Data, gives general statistic on the analyzed trajectories such as the aircraft distribution, the airport distribution, and the runways QFU distributions. Figure 2 gives an example of the section Data. The second section, called Statistics, illustrated in Figure 3, is dedicated to operational statistics of the analyzed trajectories. It gives the distribution of the analyzed trajectories in terms of compliance [START_REF] Jarry | Aircraft atypical approach detection using functional principal component analysis[END_REF], operational limits [START_REF] Jarry | Aircraft atypical approach detection using functional principal component analysis[END_REF], atypicality, event phase, event intensity level, and event number per flights.

The third section, called Events, represented in Figure 4, displays for each type of event, its number of occurrences and intensity. It provides a list of the event type sorted by their number and intensity.

Finally, the last section, called Flight Study, represented in Figure 5, enables analyzing individually each flight. It gives the different properties of the flight, the compliance, the operational limit, the atypicality per area, and the events notified for this flight. It also displays different parameters.

There are four graphs displayed in the Flight Study section. The first graph shows the longitudinal trajectory path and is illustrated in Figure 6. Besides, the user is able to display compliance limits. For instance, the flight illustrated in figure 6 presents a non-compliance corresponding to a lateral deviation [START_REF] Jarry | Aircraft atypical approach detection using functional principal component analysis[END_REF] for the 4000ft FAP procedure at Paris Orly runway 26.

The second graph, represented in figure 7, illustrates the altitude profile and the atypical coefficients represented by the colored dots. The atypical coefficient is bounded between 0 (corresponding to nominal situations and displayed in green), and 1 (corresponding to atypical situations displayed in red). The user is also able to display operational or compliance limits. For instance, the flight illustrated in figure 7 presents a glide deviation associated with the event Glide Interception From Above [START_REF] Jarry | Aircraft atypical approach detection using functional principal component analysis[END_REF]. The atypical coefficient highlights this atypical area.

The third graph, illustrated in Figure 8, shows the ground speed profile, the computed airspeed profile, and the atypical coefficients. Operational limits are also available. The flight illustrated in figure 8 presents a late speed reduction associated with an atypical area in red. Finally, the fourth graph, represented in figure 9, illustrates the aircraft engine power, and aircraft configuration elements. The blue horizontal bar shows the use of speed brakes. The vertical dashed lines display the flaps and landing gear sequence. Several Gaussian curves are also represented. They illustrate the distribution of flaps and landing gear sequence over all the flights available for this QFU. This enables analyzing this particular flight flaps configuration policy compared with the others.

B. Software group by interaction

The user is able to use TAPAS interactions to group the trajectories regarding different parameters. This enables showing statistics over a particular trajectory set, highlighting correlations, and studying a particular trajectory group. The user is able to select compliant, non-compliant, nominal, warning, critical, typical and atypical trajectories [START_REF] Jarry | Aircraft atypical approach detection using functional principal component analysis[END_REF]. In addition, the user can select a particular airport, runway QFU, aircraft type, and the phase of the atypicality (25NM-15NM, 15NM-5NM, 5NM-THR).

V. RESULTS

A. Validation and Studies

This section aims at validating the atypical event detection algorithm and showing the relevance of the use of this new process. This study analyses 14864 different A320 approach trajectories at the airports' runway QFU detailed in Table I 1) Correlation: Typical flights are compared with atypical flights, and several strong correlations are revealed. First, the compliance highly decreases, from 73.6% for typical flights to 53.2% for atypical flights. In addition, while the ratio of flight over-passing the operational critical limits [START_REF] Jarry | Aircraft atypical approach detection using functional principal component analysis[END_REF] is 5.5% for typical flights, it is 56.6% for atypical flights. These first two figures highlight the accurate behavior of the detection algorithm. Indeed, the atypical flights detected include a nonnegligible number of flights with operational issues regarding the compliance criteria and critical operational limits. Second, other correlations are found with the airline's safety office events. Regarding the number of events notified per flight, typical flights present 27.8% of flights without any events and 5.4% of flights with more than three safety events. At the opposite, atypical flights include only 9.7% of flights without any events, and 30.6% of flights with more than three safety events. Besides, the event intensity level is also correlated. Typical flights present only 18.5 high-intensity events per 100 flights on average, while there are 92.7 high-intensity events per 100 flight in average for atypical flights. It shows that atypical flights are strongly correlated with safety events.

In addition, the atypicality location is studied. The study range (25NM to the runway threshold) is divided into three phases: 25NM to 15NM, 15NM to 5NM and 5NM to the runway threshold. Airline safety offices mainly focus on the last phase (5NM to the runway threshold), which corresponds to the stabilization phase. The above behavior is confirmed with a strong correlation between the atypicality location phase and the numbze of flight safety events and their intensity. Focusing on atypical flights between 25NM to 15NM, there are 15.8% of flights with more than 3 safety events. The atypical flights between 5NM and the runway threshold present 54.0% of flights with more than 3 safety events.

2) Atypicality Appearance Phase: The location study leads to interesting atypicality appearance phase statistics detailed in Figure 10. There are three trends. First, atypicalities appearing during the 25NM to 15NM phase are still present during the 15NM to 5NM phase but are mainly dissipated in the 5NM to threshold (THR) phase. It seems that these kinds of energetical atypicalities are taken into consideration and appropriate energy managements followed. An example is displayed on Figure 11, where the aircraft was too high on the glide path before recovering (around 10NM). Nevertheless, this raises the question of the origin of the atypicality. Second, atypicalities appearing during the 15NM to 5NM phase are half dissipated in the 5NM to THR but still represent 37% of atypicalities in the 5NM to THR phase. Figure 7 shows an example of this kind of trajectories. The aircraft over-shooted the glide path. It recovered the nominal path just before stabilization. This group of atypicality presents a major interest. Indeed they may induce severe safety events but could be reduced if decisions are taken when detected. Finally, atypicality appearing during the 5NM to THR phase are critical. They are mainly due to late speed reductions, tailwind or glide path deviations. An example of late speed reduction is displayed in Figure 8. The explanations might be ATC speed constraints, pilot reduction policies, or even exterior factors in case of a tailwind.

3) Inappropriate Control Inputs: Inappropriate control inputs are actions that do not correspond to those recommended by the Standard Operating Procedure (SOP) documentation. In this section, actions relative to landing configuration are studied and correlated with atypicality.

a) Landing Configuration Time: Figure 12 represents the landing configuration time distribution (left axis) and atypical ratio for each time group (right axis). It corresponds to the time spent between the first and the last element. The average landing configuration time is between two and three minutes, while the fastest is 24 seconds. Short landing configuration times might induce high workloads for pilots. The atypical ratio was computed per minutes group (less than one minutes, between one and two minutes etc.). It is interesting to notice that the atypical ratio increases to 16.1% for the less than one minute group. This group is mainly composed of fast approaches or late reduction flights.

b) Landing gear down setting in the landing sequence: Figure 13 analyses the landing gear down setting appearance distribution in the landing configuration sequence correlated with the atypical ratio per category. For the A320, it is recommended to apply gear down while flaps 2 configuration is out. This is well described with the distribution in Figure 13 since this is what a great majority of flight did. However, a non-negligible number of flights applied gear down while being flaps 1 or flaps 0. The correlation with the atypical ratio is meaningful with 9.8% for the flaps 1 group and 29.6% the for flaps 0 group. It describes underlying energy managements. Indeed it mainly corresponds to atypicality in the 25NM to 15NM phase, and pilots anticipated landing gear configuration in order to dissipate an excess of energy. c) Last landing configuration element distance to the mean: Figure 14 represents the distance in nautical miles to the mean last landing configuration element correlated with the atypical ratio. It is similar to the Gaussian curves displayed in Figure 9 aggregated for all the QFUs. Atypical ratios were computed per nautical mile groups. When the last landing configuration element appears between 1NM and 2NM after the mean distance, the atypical ration increases up to 6.2%, and up to 27.7% between 2NM and 3NM. The group is composed of late configurations and usually late power reductions. This paper presents TAPAS, a post-operational aircraft approach analysis tool. This software uses an existing atypical flight detection algorithm using Functional Principal Component analysis to analyze aircraft approach energy management. The software was used to validate the algorithm on flight data record and flight safety office events. The software design and interactions enable relevant correlation and analysis. In particular, the atypicality phase of appearance and inappropriate control input raise interesting safety discussions and possible safety enhancement policies.

Future works will focus on extending this analysis to other aircraft types such as B737. Other studies will be done on ATC speed constraints and atypical ratio correlation. A realtime extension of the algorithm is currently being developed. In addition, discussions have begun with FDM software providers to integrate TAPAS into an energy management module of an FDM analysis software.

Figure 1 :

 1 Figure 1: This figure illustrates the atypical detection methodology process

Figure 2 :

 2 Figure 2: The Data section of TAPAS giving general information of the data.

Figure 3 :

 3 Figure 3: The Data section of TAPAS giving operational statistics of the data

Figure 4 :

 4 Figure 4: The Event section of TAPAS providing flight safety event statistics.

Figure 5 :

 5 Figure 5: The Flight Study section of TAPAS enabling single flight approach analysis

Figure 6 :

 6 Figure 6: The first graph of TAPAS section Flight Study, displaying the longitudinal path and lateral compliance criteria

Figure 7 :

 7 Figure 7: The second graph of TAPAS section Flight Study, displaying the altitude profile. The colored dots corresponds to the atypicality coefficients between nominal (0 and green) and atypical (1 and red).

Figure 8 :

 8 Figure 8: The third graph of TAPAS section Flight Study, displaying the ground speed and computed air speed profile. The colored dots corresponds to the atypical coefficients between nominal (0 and green) and atypical (1 and red).

Figure 9 :

 9 Figure 9: The fourth graph of TAPAS section Flight Study, displaying the engine power, the flaps settings, the landing gear setting, and the airbrake use. (The Gaussian curves illustrate the distribution of configuration elements for all the flights with this specific runway QFU)

Figure 10 :

 10 Figure 10: An illustration of the atypicality appearance phase

Figure 11 :

 11 Figure 11: An example of an atyical flight during phase 25NM to 15NM and 15NM to 5NM

Figure 12 :Figure 13 :

 1213 Figure 12: This figures illustrates landing configuration time distribution and atypical ratio correlation for all the A320 flights

Figure 14 :

 14 Figure 14: This figures shows last landing configuration element distance to the mean distribution and the atypical ratio correlation for all the A320 flights

TABLE I :

 I THIS

  TABLE SUMS UP THE AIRPORTS RUNWAY QFUS ANALYSED

	IN THE STUDY
	Airport Name -OACI Index	QFU
	Béjaïa -DAAE	26
		09
	Alger -DAAG	23
		27
	Tlemcen -DAON	25
		06
	Paris Orly -LFPO	08
		26
		17L
	Lyon Saint Exupery	17R
	LFLL	35L
		35R
		13L
	Marseille Provence-LFML	31L
		31R

ACKNOWLEDGMENT

The authors would like to show their gratitude to Aigle-Azur airline company for providing Flight Data Monitoring records.