Moufidath Adjibade

Camille Davisse-Paturet

Amandine Divaret-Chauveau

Karine Adel-Patient

Chantal Raherison

Marie-Noëlle Dufourg

Sandrine Lioret

Marie-Aline Charles

Blandine De Lauzon-Guillain

Enrichment of Formula in Probiotics or Prebiotics and Risk of Infection and Allergic Diseases up to Age 5.5 Years in the Nationwide Etude Longitudinale Française depuis l'Enfance (ELFE) Cohort

Keywords: Infancy, Enrichment, Infant formula, Prebiotic, Probiotic, Infection, Allergy, Birth cohort

come

Sources of Support:

The present study is funded by an ANR grant (InfaDiet project, grant number: ANR-19-CE36-0008). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Conflict of interest:

Amandine Divaret-Chauveau has been invited to allergology and pediatric congresses Results: At age 2 months, more than half of formula-fed infants consumed probiotic-enriched formula and only one in 10 consumed prebiotic-enriched formula. Consumption of Bifidobacterium lactis-enriched formula at 2 months was associated with a lower risk of LRTI (OR[95%CI]=0.84[0.73-0.96]). Consumption of Bifidobacterium breve-enriched formula up to 6 months was associated with a higher risk of LRTI (OR[95%CI]=1.75 [1.29-2.38]) and asthma (OR[95%CI]=1.95[1.28-2.97]), while its consumption from 6-10 months was associated with a lower risk of LRTI (OR[95%CI]=0.64[0.48-0.86]) and asthma (OR[95%CI]=0.59[0.40-0.88]). Moreover, consumption of Streptococcus thermophilus from 6-10 months was associated with a higher risk of asthma (OR[95%CI]=1.84 [1.29-2.63]). No significant association was found for gastrointestinal infection, food allergy, and itchy rash.

INTRODUCTION

There is increasing evidence that the microbiome could influence the infant's immune system development and that an imbalanced microbial composition and reduced diversity (dysbiosis) could promote susceptibility to metabolic and immune-related diseases [START_REF] Mortensen | Modeling transfer of vaginal microbiota from mother to infant in early life[END_REF][START_REF] Van Best | On the origin of species: Factors shaping the establishment of infant's gut microbiota[END_REF]. Colonization of the digestive tract and formation of the newborn's microbiota begins mainly at birth, and both its composition and function are variable for the first 3 years of life, after which it stabilizes [START_REF] Van Best | On the origin of species: Factors shaping the establishment of infant's gut microbiota[END_REF][START_REF] Stewart | Temporal development of the gut microbiome in early childhood from the TEDDY study[END_REF]. The infant gut microbiome composition is affected by several factors such as the maternal microbiota composition, mode of delivery, feeding practices, environmental exposures and antibiotics use [START_REF] Van Best | On the origin of species: Factors shaping the establishment of infant's gut microbiota[END_REF][START_REF] Cukrowska | The Relationship between the Infant Gut Microbiota and Allergy. The Role of Bifidobacterium breve and Prebiotic Oligosaccharides in the Activation of Anti-Allergic Mechanisms in Early Life[END_REF][START_REF] Pannaraj | Association Between Breast Milk Bacterial Communities and Establishment and Development of the Infant Gut Microbiome[END_REF][START_REF] Thompson | Milk-and solid-feeding practices and daycare attendance are associated with differences in bacterial diversity, predominant communities, and metabolic and immune function of the infant gut microbiome[END_REF][START_REF] Moore | Temporal development of the infant gut microbiome[END_REF].

Breastfeeding is the preferred nutrition option for newborns because it provides all of their nutritional needs [START_REF] Who | Feeding and nutrition of infants and young children, guidelines for the WHO European region, with emphasis on the former Soviet countries[END_REF]. Human breast milk is a complex biofluid that contains proteins, lipids, carbohydrates and various minerals and vitamins [START_REF] Moore | Temporal development of the infant gut microbiome[END_REF][START_REF] Richard | Evidence for the essentiality of arachidonic and docosahexaenoic acid in the postnatal maternal and infant diet for the development of the infant's immune system early in life[END_REF]. It also contains a wide range of nonnutritional bioactive components and a variety of both prebiotics and probiotics that ensure neonate gut colonization by microbes beneficial for metabolism, immune development and lifelong health [START_REF] Mortensen | Modeling transfer of vaginal microbiota from mother to infant in early life[END_REF][START_REF] Pannaraj | Association Between Breast Milk Bacterial Communities and Establishment and Development of the Infant Gut Microbiome[END_REF][START_REF] Doare | Mother's Milk: A Purposeful Contribution to the Development of the Infant Microbiota and Immunity[END_REF]. According to the Food and Agriculture Organization and the World Health Organization, probiotics can be considered "living microorganisms, which when administered in adequate amounts confer health benefits on the host", while prebiotics are defined as "non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacterial species already established in the colon, and thus in effect improve host health" [START_REF] Fao | Probiotics in food: Health and nutrition properties of probiotics in food including powder milk with live lactic acid bacteria and guidelines for the evaluation[END_REF].

Although exclusive breastfeeding for the first 6 months of life or at least 4 months is the nutritional recommendation for healthy term infants (8), several studies of infant feeding practices have shown a high level of non-compliance with these recommendations [START_REF] Victora | Breastfeeding in the 21st century: epidemiology, mechanisms, and lifelong effect[END_REF][START_REF] Schiess | Introduction of complementary feeding in 5 European countries[END_REF][START_REF] Cai | Global trends in exclusive breastfeeding[END_REF].

When exclusive breastfeeding is not attainable, infant formula is the preferred option. To reproduce some of the beneficial aspects of breastmilk, a large number of formulas are enriched with probiotics, prebiotics, or both (i.e., symbiotics) [START_REF] Vandenplas | Prebiotics in infant formula[END_REF][START_REF] Federik | Use of probiotic, prebiotic and symbiotic in infant formulas[END_REF].

Several randomized controlled trials (RCTs) have assessed the effect of probiotic-, prebioticor symbiotic-enriched formula on infant health. Some showed a beneficial effect of probiotics and prebiotics on respiratory or gastrointestinal infections, antibiotics use, atopic dermatitis or other types of allergies [START_REF] Maldonado | Safety and tolerance of the human milk probiotic strain Lactobacillus salivarius CECT5713 in 6-month-old children[END_REF][START_REF] Weizman | Effect of a probiotic infant formula on infections in child care centers: comparison of two probiotic agents[END_REF][START_REF] Rautava | Specific probiotics in reducing the risk of acute infections in infancy--a randomised, double-blind, placebo-controlled study[END_REF][START_REF] Bruzzese | A formula containing galacto-and fructooligosaccharides prevents intestinal and extra-intestinal infections: an observational study[END_REF][START_REF] Arslanoglu | Early dietary intervention with a mixture of prebiotic oligosaccharides reduces the incidence of allergic manifestations and infections during the first two years of life[END_REF][START_REF] Cuello-Garcia | Prebiotics for the prevention of allergies: A systematic review and meta-analysis of randomized controlled trials[END_REF], but others did not report any association with incidence of diarrhoea episodes, respiratory illnesses and allergic manifestations [START_REF] Weizman | Effect of a probiotic infant formula on infections in child care centers: comparison of two probiotic agents[END_REF][START_REF] Rautava | Specific probiotics in reducing the risk of acute infections in infancy--a randomised, double-blind, placebo-controlled study[END_REF][START_REF] Bruzzese | A formula containing galacto-and fructooligosaccharides prevents intestinal and extra-intestinal infections: an observational study[END_REF][START_REF] Arslanoglu | Early dietary intervention with a mixture of prebiotic oligosaccharides reduces the incidence of allergic manifestations and infections during the first two years of life[END_REF][START_REF] Cuello-Garcia | Prebiotics for the prevention of allergies: A systematic review and meta-analysis of randomized controlled trials[END_REF][START_REF] Sierra | Prebiotic effect during the first year of life in healthy infants fed formula containing GOS as the only prebiotic: a multicentre, randomised, doubleblind and placebo-controlled trial[END_REF][START_REF] Thibault | Effects of long-term consumption of a fermented infant formula (with Bifidobacterium breve c50 and Streptococcus thermophilus 065) on acute diarrhea in healthy infants[END_REF]. Moreover, systematic reviews concluded that evidence was not conclusive for recommending the routine use of probiotic-, prebiotic-or symbiotic-enriched formula in healthy term infants [START_REF] Skórka | To add or not to add probiotics to infant formulae? An updated systematic review[END_REF][START_REF] Skórka | Infant formulae supplemented with prebiotics: Are they better than unsupplemented formulae? An updated systematic review[END_REF][START_REF] Mugambi | Synbiotics, probiotics or prebiotics in infant formula for full term infants: a systematic review[END_REF][START_REF] De Silva | Preventing food allergy in infancy and childhood: Systematic review of randomised controlled trials[END_REF].

The Committee on Nutrition from the European Society for Pediatric Gastroenterology, Hepatology and Nutrition also concluded lack of data to recommend their routine use [START_REF] Braegger | Supplementation of infant formula with probiotics and/or prebiotics: a systematic review and comment by the ESPGHAN committee on nutrition[END_REF].

In this study, we aimed to describe the consumption of probiotic-or prebiotic-enriched formula and its association with risk of infection and allergic diseases in early childhood.

SUBJECTS AND METHODS

Study design

This analysis was based on data from the Etude Longitudinale Française depuis l'Enfance (ELFE) study, a multidisciplinary nationwide birth cohort including 18,329 children born in 2011 in 320 participating maternity units among a random sample of 349 maternities in mainland France [START_REF] Charles | Cohort Profile: The French national cohort of children (ELFE): birth to 5 years[END_REF]. Inclusion began in April 2011 and took place during 25 selected recruitment days over four waves of 4 to 8days each that covered all four seasons. Inclusion criteria were singleton or twins born after 33 weeks' gestation, from mothers aged 18 years or older and not planning to move outside of metropolitan France in the next 3 years. Foreign families also participated in the study if mothers were able to read French, Arabic, Turkish or English.

Participating mothers had to provide written consent for their own and their child's participation. Fathers signed the consent form for the child's participation when present at inclusion or were informed about their rights to oppose it. The

Data collection

Families were followed by phone interview at age 2 months, 1 year, 2 years, 3.5 years and 5.5 years. They also completed a monthly internet/paper-based questionnaire on infant feeding from age 3 to 10 months.

Infant milk feeding

At the 2-month interview, if relevant, the age at first introduction of infant formula was collected.

From age 2 to 10 months, the name and brand of the formula used was collected monthly for formula-fed infants. Classification of all reported infant formulas was based on their nutritional characteristics (ingredient list and nutritional composition [GOSs] and fructo-oligosaccharides [FOSs]) was identified from the ingredient list.

Infants receiving both breast milk and formula were classified according to the type of formula they received.

Infections and allergies

In this study, severe/frequent infections, as well as allergies from age 2 months to 5.5 years were assessed as follows:

-Ever gastrointestinal infection: parental report of at least one hospitalization for gastroenteritis/dehydration or emergency consultation for diarrhea/vomiting/dehydration -Ever upper respiratory tract infection (URTI): parental report of at least one hospitalization for URTI or emergency consultation for otalgia, at least three otitis events from birth, at least three laryngitis events from birth, or at least three angina events from birth -Ever lower respiratory tract infection (LRTI): parental report of at least one bronchiolitis event or one hospitalization for bronchitis/bronchiolitis/pneumopathy -Ever wheezing, asthma, itchy rash or food allergy: at least one parental report of wheezing in the chest, medical diagnosis of asthma, itchy rash, or medical advice to avoid certain foods due to a food allergy.

Other variables

Parental socio-demographic characteristics considered in this study were: maternal age at delivery (18-24, 25-29, 30-34, ≥35 years), number of older children in the household (no sibling, one sibling, at least two siblings), maternal migration status ("majority population" which included women born to two French parents [inside or outside of France]), "descendants of migrants" including women born in France with at least one non-French parent, "migrants" including women not born in France and without French citizenship at birth), maternal education level (up to lower secondary, upper secondary, intermediate, 3-year university degree, at least 5-year university degree), employment status during pregnancy (employed, unemployed, not in the labour force [e.g., housewife, student, disabled, retired]) and monthly household income per consumption unit (<€750, €751-1,111, €1,112-1,500, €1,501-1,944, €1,945-2,500, >€2,500). From the postal code of residence, we determined the region of residence (Paris region, North, East, East Paris Basin, West Paris Basin, West, South-West, South-East, Mediterranean) and the city size (rural area, urban area; >2000 inhabitants for rural area).

Maternal health characteristics included self-reported height and pre-pregnancy weight used to calculate pre-pregnancy body mass index (BMI) (<18.5, 18.5-24.9, 25.0-29.9, ≥30.0 kg/m²), smoking status (never smoker, smoker only before pregnancy, smoker only in early pregnancy, smoker throughout pregnancy) and diet quality during pregnancy (measured by using the Probability of Adequate Nutrient intake based Diet quality index [PANDiet] score, which reflects nutrient-based reference guidelines adapted for pregnancy; total scores range from 0 to 100) [START_REF] Bianchi | Pregnancy Requires Major Changes in the Quality of the Diet for Nutritional Adequacy: Simulations in the French and the United States Populations[END_REF]. Newborn characteristics were collected from the medical record: child sex, gestational age, birth weight and delivery mode (C-section, vaginal). Birth weight categories (small, adequate or large for gestational age) were defined according to Audipog reference curves [START_REF] Mamelle | Fetal growth from the AUDIPOG study. I. Establishment of reference curves[END_REF]. The 2month questionnaire collected the type of the physician consulted for the first visit after delivery (paediatrician, other child doctor, general practitioner, other including emergency).

Parent and sibling history of allergy (hay fever, asthma, eczema) was collected at the 2-month interview. Children were considered to have a family history of allergy if at least one parent or sibling had a history of allergy.

Study sample

Children whose parents withdrew their consent within the first year and requested deletion of their data (n=57) were excluded from this study. We also randomly excluded one twin out of two (n=287) to avoid family clustering.

For the present analysis, children not followed at the 2-month interview (n=1,696), those exclusively breastfed at age 2 months (n=5,045), those without sufficient data on the infant formula consumed at age 2 months (n=667), those with a medical diagnosis of cow's milk protein allergy at age 2 months (n=193) or those without any data on infection and allergy from age 2 months to 5.5 years (n=1,995) were excluded. The main analyses then involved 8,389 children (Figure 1).

Statistical analyses

Descriptive statistics

Differences between excluded children (n=9,596) and children included in the main analyses (n=8,389) were assessed with Student t test and chi-square test for continuous and categorical variables, respectively.

To take into account the inclusion procedure and biases related to non-consent and attrition, the data were weighted [START_REF] Juillard | Weighting of Elfe survey data at time 0. pandora[END_REF] in sensitivity analyses. Weighting also included calibration on margins from the state register's statistical data and the 2010 French National Perinatal study [START_REF] Blondel | Trends in perinatal health in France from 1995 to 2010. Results from the French National Perinatal Surveys[END_REF] for the following variables: age, region, marital status, migration status, education level, and primiparity.

Main analyses

Multiple imputations were performed to deal with missing data (covariates and longitudinal trajectories of the type of formula consumed from age 2 to 10 months) [START_REF] Sterne | Multiple imputation for missing data in epidemiological and clinical research: potential and pitfalls[END_REF][START_REF] Van Buuren | Multiple imputation of discrete and continuous data by fully conditional specification[END_REF]. We assumed that data were missing at random, i.e. that missing values in the data set may depend on the value of other observed variables, but cannot depend on the values of unobserved variables [START_REF] Briggs | Missing... presumed at random: costanalysis of incomplete data[END_REF]. We generated five independent data sets by using the fully conditional specification method (MI procedure) and the calculated estimates of pooled effects (MIANALYSE procedure; SAS software). Missing data for categorical variables were imputed by using a multinomial model, for ordinal or binary variables by using logistic regression and for the continuous variables by using linear regression [START_REF] Van Buuren | Multiple imputation of discrete and continuous data by fully conditional specification[END_REF].

Adjusted associations between consumption of enriched formula and risk of infection or allergic diseases were examined by logistic regression models adjusted for confounding factors. Potential confounding factors were identified first from the literature, then selected by using the directed acyclic graphs method [START_REF] Shrier | Reducing bias through directed acyclic graphs[END_REF]. The main model was adjusted for maternal and household characteristics (maternal age, education level, migration status, employment during pregnancy, smoking status, city size, region of residence, maternal diet quality during pregnancy [PANDiet score], number of older children, household income and family history of allergy), child characteristics (sex, gestational age, birth weight category, type of physician consulted after discharge, C-section delivery, age at infant formula introduction) and variables related to study design (maternity size and recruitment wave). A second model was performed to additionally account for the use of antibiotics up to age 1 year. Formula enriched with probiotics or prebiotics was first examined at the 2-month follow-up (enriched vs not enriched). Then, to account for the temporal evolution in the consumption of prebiotic or probiotic-enriched formula between child age 2 and 10 months, we identified groups of children with similar longitudinal patterns of consumption by using the Nagin's method for group-based trajectory modelling (GBTM) [START_REF] Nagin | Group-Based Modeling of Development[END_REF] with the TRAJ procedure from SAS software. These trajectories were modeled for children who consumed formula for at least 2 follow-ups from age 2 to 10 months.

Sensitivity analyses

Sensitivity analyses were conducted for children whose parents did not report any eczema, wheezing, gastrointestinal infection or respiratory tract infection at age 2 months (n=6,502), to address potential reverse causation bias.

Additional sensitivity analyses involved exclusively formula-fed infants at age 2 months (n=6,251). Moreover, to deal with the issue of change in infant formula from birth to the 2month interview, a sensitivity analysis involved children without any change in infant formula up to age 2 months (n=4,399). Finally, given our previous findings on the association between partially hydrolyzed formula and allergy [START_REF] Davisse-Paturet | Use of partially hydrolysed formula in infancy and incidence of eczema, respiratory symptoms or food allergies in toddlers from the ELFE cohort[END_REF], a sensitivity analysis was conducted among infants consuming non-hydrolyzed formula (n=7,805).

To deal with selection and attrition bias, a sensitivity analysis was conducted with weighted data according to the weighting described previously. Finally, as the main analyses used multiple imputation to deal with missing data, the main analyses were replicated on the complete-case sample.

All analyses were carried out with SAS 9.4 (SAS Institute, Cary, NC, USA).

RESULTS

As compared with excluded children, included children frequently were the first born (46.6% vs 42.3%, p<0.0001), were born to older mothers (mean age 31.0 ± 4.9 vs 30.6 ± 5.2 years, p=0.00003), mothers who were employed during pregnancy (75.2% vs 67.3%, p<0.0001) and had a higher education level (40.8% vs. [START_REF] Sterne | Multiple imputation for missing data in epidemiological and clinical research: potential and pitfalls[END_REF].9% with at least 3-year university degree, p<0.0001). The characteristics of included children are described in Table 1.

Enrichment of formulas with prebiotics and probiotics

At the 2-month follow-up, more than half of formula-fed infants consumed probiotic-enriched formula. The enrichment with prebiotics usually using a mixture of GOS and FOS, concerned 9.1% (95% confidence interval=8.4%-9.8%) of infants.

The weighted prevalence of formula-fed infants consuming probiotic-enriched formula at the 2-month follow-up is presented in Figure 2. The main probiotics added to infant formula were Streptococcus thermophilus, Lactobacillus reuteri (DSM 17938), and Bifidobacterium breve (BC50), followed by Lactobacillus fermentum (CECT5716) and Bifidobacterium lactis (BB12). Enrichment with S. thermophilus was always associated with another enrichment (B. breve, B. lactis or L. reuteri). The combined enrichment with Bifidobacterium infantis and Lactobacillus rhamnosus was consumed by less than 3% of infants and was not further examined.

The longitudinal trajectories of consumption of formulas enriched with probiotics or prebiotics from age 2 to 10 months are presented in Supplementary Figure 1. For B. breve and S. thermophilus-enriched formula, a 4-group solution was identified with infants consuming 1) never-enriched formula, 2) enriched formula up to age 5-6 months, 3) enriched formula from age 6-7 months and 4) enriched formula throughout infancy. For L. fermentum and L. reuteri-enriched formula, a 3-group solution was identified with infants consuming 1) never-enriched formula, 2) occasionally-enriched formula and 3) enriched formula throughout infancy. For B. lactis-enriched formula, a 2-group solution was identified with infants consuming 1) never-enriched formula and 2) enriched formula throughout infancy. For GOSenriched formulas, a 3-group solution was identified, with infants consuming 1) neverenriched formula, 2) early use of enriched formula and 3) delayed use of enriched formula.

Probiotic-enriched formula and risk of infection or allergic diseases up to age 5.5 years

The findings of unadjusted analyses are presented in Supplemental tables 1 and 2.

Bifidobacterium enrichment

In the main model, compared to infants consuming non-Bifidobacterium-enriched formula, those consuming B. lactis-enriched formula at age 2 months were at lower risk of LRTI and wheezing from age 2 months to 5.5 years (Table 2). We found no significant association between Bifidobacterium enrichment and URTI, asthma (Table 2), gastrointestinal infection, food allergy or itchy rash up to age 5.5 years (Table 3). Further adjustment for antibiotic use up to age 1 year did not substantially modify the risk estimates, but the observed associations between consumption of B. lactis-enriched formula and wheezing were no longer significant (data not shown).

The observed associations for the consumption of Bifidobacterium-enriched formula and the risk of respiratory symptoms, gastrointestinal infection, food allergy and itchy rash were consistent in analyses among exclusively formula-fed infants (Supplementary tables 3 and 4), in weighted analyses and analyses in the complete-case sample (Supplementary tables 5-8). Similar findings were also observed in the other sensitivity analyses (analyses conducted in subsamples of infants without any symptoms up to 2 months, infants without any change in formula up to age 2 months (Supplementary tables 9-12), and infants consuming nonhydrolyzed formula (data not shown).

According to the GBTM method, infants always consuming B. lactis-enriched formula from age 2 to 10 months were also at lower risk of URTI and asthma up to age 5.5 years (Table 2).

Consumption from age 6 months of B. breve-enriched formula was related to a lower risk of LRTI and asthma up to age 5.5 years, whereas consumption in the first months only was related to a higher risk of LRTI and asthma up to age 5.5 years (Table 2). Similar findings were observed after further adjustment for antibiotic use (data not shown) and in sensitivity analyses (Supplementary tables 3-12).

Lactobacillus enrichment

In the main model, compared to consumption of non-Lactobacillus-enriched formula, consumption of L. reuteri-enriched or L. fermentum-enriched formula at age 2 months was not associated with occurrence of infection or allergic diseases up to age 5.5 years (Tables 2 and3). After further adjustment for antibiotic use, consumption of L. reuteri-enriched formula at age 2 months was related to a lower risk of URTI up to age 5.5 years (OR=0.83 and 95% CI=0.72-0.97; data not shown).

According to the GBTM method, occasional, or regular consumption of L. reuteri-enriched formula from age 2 to 10 months was not associated with occurrence of infection or allergic diseases (Tables 2 and3). Occasional consumption of L. fermentum-enriched formula from age 2 to 10 months was related to a higher risk of URTI up to age 5.5 years, but not regular consumption. Consumption of L. fermentum-enriched formula was not related to other infections or allergic diseases. Similar findings were observed after further adjustment for antibiotic use (data not shown) and in sensitivity analyses (Supplementary tables 3-12).

Streptococcus thermophilus enrichment

In the main model, compared to consumption of non-S. thermophilus-enriched formula at age 2 months, consumption of S. thermophilus-enriched formula was not related to infections or allergic diseases up to age 5.5 years (Tables 2 and3). Similar findings were observed after further adjustment for antibiotic use (data not shown).

According the GBTM method, consumption of S. thermophilus-enriched formula from age 2 to 10 months was related to a higher risk of URTI up to age 5.5 years. In addition, consumption of S. thermophilus-enriched formula up to age 6 months was related to a lower risk of asthma up to age 5.5 years, whereas consumption from 6 months was related to a higher risk of asthma (Table 2). Consumption of S. thermophilus-enriched formula was not related to other infections or allergic diseases (Tables 2 and3). Similar findings were observed after further adjustment for antibiotic use (data not shown) and in sensitivity analyses (Supplementary tables 3-12).

Prebiotic-enriched formula and risk of infection or allergic diseases up to age 5.5 years

Consumption of prebiotic-enriched formula at age 2 months or between 2 and 10 months was not related to infection and allergic diseases, except for the association between early use of GOS-enriched formula and a lower risk of URTI (Tables 2 and3). However, this association was no longer significant after further adjustment for antibiotic use up to age 1 year (data not shown). The observed findings for the consumption of prebiotic-enriched formula and the risk of infection and allergic diseases were similar in sensitivity analyses (Supplementary tables 3-12).

DISCUSSION

Among children born in 2011, more than half of formula-fed infants had consumed probioticenriched formula at age 2 months. The infant formula consumed was often enriched with L. reuteri (DSM 17938) or B. breve (BC50) combined with S. thermophilus, followed by L. fermentum (CECT5716) and B. lactis (BB12) combined with S. thermophilus. Overall, the consumption of probiotic-enriched formula was related to the risk of respiratory diseases (respiratory tract infection, wheezing, asthma) up to age 5.5 years, but not with gastrointestinal infection, food allergy, and itchy rash. However, the observed associations for respiratory diseases were not consistent depending on the strains used for enrichment, as well as the consumption period of the formula. No significant association was found for the consumption of prebiotic-enriched formula.

As in our study, most RCTs highlighted null or beneficial effect of probiotic enrichment on respiratory diseases or respiratory infection-related outcomes [START_REF] Skórka | To add or not to add probiotics to infant formulae? An updated systematic review[END_REF][START_REF] Braegger | Supplementation of infant formula with probiotics and/or prebiotics: a systematic review and comment by the ESPGHAN committee on nutrition[END_REF]. However, when considering the strains, results are less constituent. Indeed, only one RCT including 80 healthy 6-month-old children in Spain showed a lower number of episodes of respiratory infections with consumption of formula enriched with L. salivarius CECT5713, compared with non-enriched formula [START_REF] Maldonado | Safety and tolerance of the human milk probiotic strain Lactobacillus salivarius CECT5713 in 6-month-old children[END_REF]. Another RCT involving 81 healthy infants in Finland who consumed infant formula before age 2 months also showed a protective effect of formula enriched with B. lactis BB12 combined with L. rhamnosus GG ATCC53103 on the incidence of recurrent respiratory infections during the first year of life [START_REF] Rautava | Specific probiotics in reducing the risk of acute infections in infancy--a randomised, double-blind, placebo-controlled study[END_REF]. However, in this study, probiotics were not added to the formula during manufacturing, but rather as capsules (prebiotics/placebo) directly added to the formula. In addition, consumption of probioticenriched formula was not related to the incidence of respiratory infections.

In the ELFE study, consumption of probiotic-enriched formula was not associated with the risk of itchy rash and food allergy, which is consistent with the findings observed in other studies [START_REF] Braegger | Supplementation of infant formula with probiotics and/or prebiotics: a systematic review and comment by the ESPGHAN committee on nutrition[END_REF]. The consumption of probiotic-enriched formula was also not associated with gastrointestinal infection in this study. Although some RCTs showed that consumption of probiotic-enriched formula (e.g. enrichment with B. lactis BB12 alone or in combined with S. thermophilus, L.s reuteri ATCC 55730 or L. salivarius CECT5713) had a protective effect on gastrointestinal infection outcomes [START_REF] Maldonado | Safety and tolerance of the human milk probiotic strain Lactobacillus salivarius CECT5713 in 6-month-old children[END_REF][START_REF] Weizman | Effect of a probiotic infant formula on infections in child care centers: comparison of two probiotic agents[END_REF][START_REF] Skórka | To add or not to add probiotics to infant formulae? An updated systematic review[END_REF], others examining the effect of formula enriched with B. lactis BB12 combined with L. rhamnosus GG ATCC53103, B. longum BL999 combined with L. rhamnosus LPR, or B. breve C50 combined with S. thermophilus or with L. johnsonii La1 found non-significant associations [START_REF] Rautava | Specific probiotics in reducing the risk of acute infections in infancy--a randomised, double-blind, placebo-controlled study[END_REF][START_REF] Thibault | Effects of long-term consumption of a fermented infant formula (with Bifidobacterium breve c50 and Streptococcus thermophilus 065) on acute diarrhea in healthy infants[END_REF][START_REF] Skórka | To add or not to add probiotics to infant formulae? An updated systematic review[END_REF][START_REF] Braegger | Supplementation of infant formula with probiotics and/or prebiotics: a systematic review and comment by the ESPGHAN committee on nutrition[END_REF].

In the present study, the effect of probiotic enrichment was evaluated for each probiotic strain, although some formulas were enriched with a combination of strains. This methodological approach does not consider the synergistic effect of probiotics strains; however, the different strains studied were adjusted for each other in all models. Interactions between strains were also tested and were not significant in any model.

Overall, studies that evaluated the effect of probiotic-enriched formula on childhood health showed inconsistent findings. This observation could be explained by the considerable heterogeneity in the RCT designs. Indeed, the types of probiotics and strains as well as the combinations used differed across studies, while the different probiotics strains have specific properties. The measured outcomes and their definitions, duration and timing of intervention also differ across studies. Further studies with a long follow-up, large sample size and using similar methodology (same inclusion criteria, duration and timing of intervention, probiotics strains, combinations and dose used) are needed to establish the effects of probiotic-enriched formula on childhood health.

Regarding the effect of prebiotics-enriched formula consumption on infection and allergy in childhood, available studies are limited and the results are inconclusive (20-23, 26, 27, 29).

We found no significant association between prebiotic-enriched formula consumption and the risk of gastrointestinal infection, respiratory symptoms and allergic diseases, consistent with the findings observed in some RCTs [START_REF] Bruzzese | A formula containing galacto-and fructooligosaccharides prevents intestinal and extra-intestinal infections: an observational study[END_REF][START_REF] Arslanoglu | Early dietary intervention with a mixture of prebiotic oligosaccharides reduces the incidence of allergic manifestations and infections during the first two years of life[END_REF][START_REF] Sierra | Prebiotic effect during the first year of life in healthy infants fed formula containing GOS as the only prebiotic: a multicentre, randomised, doubleblind and placebo-controlled trial[END_REF]. In contrast, one RCT involving healthy term infants in Italy, from parents with a history of allergy (atopic eczema, allergic rhinitis, or asthma) found lower number of episodes of URTI during the 2-year follow-up among infants consuming hypoallergenic formula enriched with a mixture of short-chain GOS and longchain FOS than those consuming the same formula without enrichment [START_REF] Arslanoglu | Early dietary intervention with a mixture of prebiotic oligosaccharides reduces the incidence of allergic manifestations and infections during the first two years of life[END_REF]. In addition, one RCT found a significantly lower number of children with at least 1 episode of acute diarrhoea and lower number of diarrheal episodes during the 12-month follow-up in the group receiving GOS/FOS-enriched formula as compared with the control group [START_REF] Bruzzese | A formula containing galacto-and fructooligosaccharides prevents intestinal and extra-intestinal infections: an observational study[END_REF], and another found that the cumulative incidences of allergic manifestations (atopic dermatitis, recurrent wheezing, and allergic urticaria) were lower in the intervention group, than in the control group [START_REF] Arslanoglu | Early dietary intervention with a mixture of prebiotic oligosaccharides reduces the incidence of allergic manifestations and infections during the first two years of life[END_REF].

To our knowledge, no other longitudinal cohort has assessed the association between probiotic or prebiotic formula enrichment and infection/allergic manifestations, which hinders international comparison of our results.

The present prospective study involved children included in a large French birth cohort with detailed data on infant diet, which allowed us to examine among formula-fed infants and under real conditions of use, the effect of formula enrichment with probiotics or prebiotics on infection and allergic diseases in childhood. In this cohort, the number of infants exposed to probiotic-enriched formula was higher than that observed in the trials (mostly small sample sizes). This high proportion could possibly be attributable to popular beliefs about the health benefits of probiotics. However, a previous study using the ELFE cohort data showed few social and health-related factors associated with the use of prebiotic-or probiotic-enriched formula [START_REF] De Lauzon-Guillain | Use of infant formula in the ELFE study: The association with social and health-related factors[END_REF], which suggests that reverse causality bias is limited in the present study. Indeed, the authors found that only family income and breastfeeding duration were related to the use of prebiotic-or probiotic-enriched formula at age 2 months and the type of infant formula used was fairly stable between 2 and 10 months.

Although a wide range of socio-demographic and economic data was considered in the present study, other unmeasured factors such as dietary supplements containing probiotics or prebiotics consumed by mothers or infants and intake of prebiotics from complementary foods might have led to potential residual confounding. In this cohort, health data were reported by parents and not validated by medical records, which could lead to a potential measurement bias. However, the items used were derived from international ones (42) to limit this bias. In addition, repeated data on health-related outcomes in childhood limited memory bias. The various sensitivity analyses performed to examine the robustness of our results and address selection and reverse causation bias showed very similar findings, which suggests that if there were any potential biases, they should have a limited impact on our findings.

In conclusion, this observational study showed that probiotic-enriched formulas are often consumed, but few show a real health benefit. The consumption of prebiotic-enriched formula does not have convincing effects on health. Replication in other observational studies and larger well-designed RCTs are needed to confirm these results and possibly identify the most interesting strains and optimal "window of opportunity" for prebiotic and probiotic-enriched formula consumption.

  The ELFE cohort is a joint project between INED (Institut National d'Etudes Démographiques), INSERM (Institut National de la Santé et de la Recherche Médicale), EFS (Etablissement Français du Sang), InVS (Institut de Veille Sanitaire), INSEE (Institut National de la Statistique et des Etudes Economiques), the Ministry of Health (DGS, Direction Générale de la Santé), the Ministry of Environment (DGPR, Direction Générale de la Prévention des Risques), the Ministry of Health and Employment (DREES, Direction de la Recherche, des Etudes, de l'Evaluation et des Statistiques), and the CNAF (Caisse Nationale des Allocations Familiales), with the support of the Ministry of Research and CCDSHS (Comité de Concertation pour les Données en Sciences Humaines et Sociales) and the Ministry of Culture (DEPS, Département des études, de la prospective et des statistiques). As part of the RECONAI platform, the study received state funding from the ANR within the framework of the "Future Investments" program (reference: ANR-11-EQPX-0038, ANR-19-COHO-0001).

(

  inscription and accommodation) by Mead Johnson, Sodilac and Nutricia. Karine Adel-Patient has been invited by Stallergènes to speak at a conference, outside of the submitted work. None of the other authors declare any conflicts of interest.

  ELFE study was approved by the Advisory Committee for Treatment of Health Research Information (Comité Consultatif sur le Traitement des Informations pour la Recherche en Santé), the National Data Protection Authority (Commission Nationale Informatique et Libertés), and the National Statistics Council.

Table 2

 2 Adjusted associations between consumption of enriched formula at 2 months and from 2 to 10 months and the risk of respiratory diseases up to 5.5 years (n=8,389)

			Upper respiratory		Lower respiratory		Wheezing		Asthma
			tract infection		tract infection
		n	OR [95% CI]	P	OR [95% CI]	P	OR [95% CI]	P	OR [95% CI]	P
	Consumption at 2 months
	Enrichment in Bifidobacterium			0.1		0.03		0.2		0.3
	No Bifidobacterium	5,805	1.00 [Ref]		1.00 [Ref]		1.00 [Ref]		1.00 [Ref]
	Bifidobacterium breve (BC50)	1,372	1.19 [1.00; 1.41]		1.09 [0.93; 1.29]		1.06 [0.90; 1.24]		0.97 [0.77; 1.21]
	Bifidobacterium lactis (BB12)	732	0.95 [0.82; 1.10]		0.84 [0.73; 0.96]		0.87 [0.75; 0.99]		0.83 [0.68; 1.01]
	Other Bifidobacterium or unspecified strain 480	0.87 [0.71; 1.08]		1.15 [0.95; 1.40]		1.10 [0.91; 1.34]		1.25 [0.96; 1.62]
	Enrichment in Lactobacillus			0.2		0.5		0.8		0.1
	No Lactobacillus	6,043	1.00 [Ref]		1.00 [Ref]		1.00 [Ref]		1.00 [Ref]
	Lactobacillus reuteri (DSM 17938)	1,679	0.86 [0.75; 1.00]		1.09 [0.95; 1.25]		1.00 [0.87; 1.14]		0.85 [0.70; 1.02]
	Lactobacillus fermentum (CECT5716)	480	1.04 [0.87; 1.24]		0.96 [0.81; 1.15]		1.08 [0.91; 1.28]		1.19 [0.95; 1.50]
	Other Lactobacillus or unspecified strain	187	1.18 [0.87; 1.60]		0.89 [0.67; 1.20]		0.95 [0.71; 1.26]		1.06 [0.72; 1.55]
	Enrichment in Streptococcus			0.5		0.6		0.3		0.3
	No Streptococcus	6,295	1.00 [Ref]		1.00 [Ref]		1.00 [Ref]		1.00 [Ref]
	Streptococcus thermophilus	2,094	0.96 [0.86; 1.07]		0.97 [0.87; 1.08]		1.06 [0.95; 1.17]		1.08 [0.94; 1.26]
	Enrichment in prebiotics			0.6		0.9		0.5		0.7
	No prebiotics	7,456	1.00 [Ref]		1.00 [Ref]		1.00 [Ref]		1.00 [Ref]
	FOS/GOS or GOS only	933	0.98 [0.91; 1.06]		1.00 [0.93; 1.08]		1.03 [0.96; 1.11]		0.98 [0.89; 1.09]

Consumption between 2 and 10 months Enrichment in B. breve (BC50)

  OR odd ratioLogistic regressions adjusted for maternal and household characteristics (maternal age, education level, migration status, employment during pregnancy, smoking status, pre-pregnancy BMI, diet quality during pregnancy, city size, region of residence, household income, family history of allergy and number of older children), child characteristics (sex, gestational age, birth weight category, type of physician consulted after discharge, C-section delivery and age at infant formula introduction) and ELFE study design variables (maternity size and recruitment wave). For a given period, all enrichments were considered simultaneously.

	Never	7,717	1.00 [Ref]	1.00 [Ref]	1.00 [Ref]	1.00 [Ref]
	Sometimes	329	1.21 [1.02; 1.44]	1.13 [0.96; 1.34]	1.13 [0.95; 1.34]	1.15 [0.92; 1.44]
	Always	343	0.92 [0.78; 1.10]	0.90 [0.76; 1.06]	1.03 [0.87; 1.22]	0.95 [0.75; 1.20]
	Enrichment in L. reuteri (DSM 17938)		0.2	0.5	0.9	0.6
	Never	6,468	1.00 [Ref]	1.00 [Ref]	1.00 [Ref]	1.00 [Ref]
	Sometimes	590	1.00 [0.88; 1.14]	1.07 [0.94; 1.22]	1.02 [0.90; 1.15]	0.98 [0.82; 1.17]
	Always	1,331	0.94 [0.84; 1.05]	0.94 [0.85; 1.05]	1.00 [0.90; 1.10]	0.97 [0.84; 1.12]
	Enrichment in S. thermophilus		0.08	0.4	0.8	0.003
	Never	5,218	1.00 [Ref]	1.00 [Ref]	1.00 [Ref]	1.00 [Ref]
	Up to 6 months	471	0.97 [0.76; 1.25]	0.81 [0.64; 1.03]	0.97 [0.77; 1.22]	0.66 [0.47; 0.94]
	From 6 months	812	0.81 [0.61; 1.09]	1.13 [0.86; 1.50]	0.99 [0.75; 1.29]	1.84 [1.29; 2.63]
	Always	1,888	1.34 [1.06; 1.69]	1.10 [0.88; 1.37]	1.10 [0.89; 1.36]	1.15 [0.85; 1.57]
	Enrichment in GOS		0.1	0.5	0.2	0.3
	Never	7,205	1.00 [Ref]	1.00 [Ref]	1.00 [Ref]	1.00 [Ref]
	Early use	487	0.87 [0.76; 0.99]	0.96 [0.85; 1.10]	1.08 [0.95; 1.22]	1.06 [0.88; 1.28]
	Delayed use	697	1.13 [0.97; 1.32]	0.99 [0.84; 1.16]	0.87 [0.75; 1.01]	0.88 [0.69; 1.12]
			0.4	0.002	0.4	0.003
	Never	6,053	1.00 [Ref]	1.00 [Ref]	1.00 [Ref]	1.00 [Ref]
	Up to 6 months	284	1.16 [0.85; 1.60]	1.75 [1.29; 2.38]	1.28 [0.96; 1.72]	1.95 [1.28; 2.97]
	From 6 months	748	1.01 [0.74; 1.40]	0.64 [0.48; 0.86]	0.90 [0.67; 1.20]	0.59 [0.40; 0.88]
	Always	1,304	0.79 [0.62; 1.02]	0.90 [0.70; 1.15]	0.90 [0.71; 1.15]	0.74 [0.52; 1.05]
	Enrichment in B. lactis (BB12)		0.01	0.2	0.1	0.04
	Never	7,598	1.00 [Ref]	1.00 [Ref]	1.00 [Ref]	1.00 [Ref]
	Always	791	0.80 [0.68; 0.95]	0.89 [0.76; 1.05]	0.89 [0.76; 1.04]	0.78 [0.62; 0.99]
	Enrichment in L. fermentum (CECT5716)		0.05	0.3	0.03	0.3

CI confidence interval; FOS fructo-oligosaccharides; GOS galacto-oligosaccharides;

Table 3

 3 Associations between consumption of enriched formula at 2 months and from 2 to 10 months and the risk of gastrointestinal infections, food allergies or itchy rash up to 5.5 years (n=8,389)

		Gastrointestinal		Food allergy		Itchy rash
		infection
		OR [95% CI]	P	OR [95% CI]	P	OR [95% CI]	P
	Consumption at 2 months
	Enrichment in Bifidobacterium		0.3		0.4		0.7
	No Bifidobacterium	1.00 [Ref]		1.00 [Ref]		1.00 [Ref]
	Bifidobacterium breve (BC50)	0.93 [0.75; 1.17]		0.87 [0.64; 1.18]		1.07 [0.91; 1.25]
	Bifidobacterium lactis (BB12)	0.92 [0.76; 1.12]		0.91 [0.68; 1.21]		1.00 [0.88; 1.15]
	Other Bifidobacterium or unspecified strain 1.27 [0.98; 1.64]		1.01 [0.67; 1.52]		1.00 [0.82; 1.21]
	Enrichment in Lactobacillus		0.5		0.2		0.8
	No Lactobacillus	1.00 [Ref]		1.00 [Ref]		1.00 [Ref]
	Lactobacillus reuteri (DSM 17938)	1.15 [0.95; 1.39]		0.78 [0.59; 1.02]		0.95 [0.83; 1.08]
	Lactobacillus fermentum (CECT5716)	0.94 [0.72; 1.21]		0.79 [0.55; 1.12]		1.00 [0.84; 1.19]
	Other Lactobacillus or unspecified strain	0.87 [0.58; 1.29]		1.74 [1.01; 3.01]		1.11 [0.83; 1.47]
	Enrichment in Streptococcus		0.9		0.06		0.3
	No Streptococcus	1.00 [Ref]		1.00 [Ref]		1.00 [Ref]
	Streptococcus thermophilus	1.01 [0.88; 1.17]		1.21 [0.99; 1.49]		0.94 [0.85; 1.04]
	Enrichment in prebiotics		0.3		0.1		0.5
	No prebiotics	1.00 [Ref]		1.00 [Ref]		1.00 [Ref]
	FOS/GOS or GOS only	1.05 [0.95; 1.17]		0.89 [0.76; 1.04]		0.98 [0.91; 1.05]

Consumption between 2 and 10 months Enrichment in B. breve (BC50)

		0.6	0.7	0.2
	Never	1.00 [Ref]	1.00 [Ref]	1.00 [Ref]
	Up to 6 months	1.21 [0.78; 1.88]	1.20 [0.69; 2.08]	1.29 [0.96; 1.73]
	From 6 months	1.06 [0.70; 1.62]	1.02 [0.56; 1.87]	0.91 [0.69; 1.20]
	Always	0.87 [0.62; 1.24]	1.00 [0.62; 1.63]	0.84 [0.66; 1.06]
	Enrichment in B. lactis (BB12)	0.8	0.6	0.5
	Never	1.00 [Ref]	1.00 [Ref]	1.00 [Ref]
	Always	1.03 [0.82; 1.28]	1.08 [0.81; 1.44]	0.95 [0.81; 1.10]
	Enrichment in L. fermentum (CECT5716)	0.5	0.1	0.9

Acknowledgments:

We thank the scientific coordinators (B Geay, H Léridon, C Bois, JL Lanoé, X Thierry, C Zaros), IT and data managers, statisticians (M Cheminat, C Ricourt, A Candea, S de Visme), administrative and family communication staff, study technicians (C Guevel, M Zoubiri, L G L Gravier, I, Milan, R Popa) of the ELFE coordination team, as well as the families that gave their time for the study.

Author's contributions: CR, M-ND, M-A C, SL and BL-G were responsible for the development of the design and protocol of the study; MA, CD-P and BL-G performed the statistical analyses; MA wrote the paper; BLG provided methodological guidance; MA, CD-P, AD-C, KA-P, CR, M-ND, SL, M-AC and BL-G were involved in interpreting the results and editing the manuscript for important intellectual content. All authors read and approved the final manuscript.


Rami Ghorbel
email: rami.ghorbel@enis.tn

Ahmed Ktari

Nader Haddar

Microstructure and mechanical property assessment of stainless steel-clad plate joint made by hybrid SMAW-GTAW multi-pass welding process

Keywords: Stainless clad steel, Multi-pass welding, Mechanical properties, Microstructure, Transition zone, Local hardening zone Pressure Vessels, Boilers, and Pipelines

The stainless clad steel plates (SCSPs) are used in several industrial fields such as petroleum and petrochemical industries due to their low cost and good corrosion resistance. In this work, the structural integrity assessment of a welded joint, produced by a hybrid SMAW/GTAW multi-pass welding process of SCSP, was carried out through non-destructive examinations, microstructural observations and mechanical characterization tests. The non-destructive examination showed that the welded zone is free from any sign of defect. The welded joint filled with ER316L revealed the presence of δ-ferrite, which was solidified as skeletal and lathy ferrite under ferritic-austenitic (FA) solidification mode. The microstructure of the weld metal transition zone (WM-TZ) filled with ER309L exhibited the presence of martensitic laths as well as a cellular and columnar structure. It was found that the diffusion of carbon element caused the forming of a decarburized layer in the substrate heat-affected zone (S-HAZ) during the welding process. The observation also revealed the presence of type I and type II boundaries along the fusion line, which are characterized by their martensitic nature. Based on the determination of the WM-TZ dilution percentage, which is equal to 32%, the presence of the martensite was also confirmed by Schaeffler diagram. The high micro-hardness values (~ 429 HV) and the low toughness (~ 22.9J) measured respectively in vicinity of the WM-TZ also confirm the local hardening zone (LHZ) formed on the welded zone after bending tests.

reduce the amount of alloy elements like chromium (Cr) and nickel (Ni) by 70-80% [START_REF] Li C'a, Qin | Microstructures and mechanical properties of stainless-steel clad plate joint with diverse filler metals[END_REF]. Considering those advantages, the SCSP has become, in the last few years, an interesting alternative to ordinary pure stainless-steel plates in many industrial applications such as petroleum, petrochemical and pressure vessel industries [7][START_REF] An | Microstructure and mechanical properties of stainless-steel clad plate joints produced by TIG and MAG hybrid welding[END_REF][9].

Several welding processes, such as shield metal arc welding (SMAW) [START_REF] Dhib | Cladding of low-carbon steel to austenitic stainless steel by hot-roll bonding: microstructure and mechanical properties before and after welding[END_REF][START_REF] Tian | Welding process of composite plate for construction and its welded microstructure and properties[END_REF], submerged arc welding (SAW) [START_REF] Qiu | Analysis on welded joint properties of stainless clad steel plates[END_REF], gas tungsten arc welding (GTAW) [START_REF] Li | Welding L415/316L Bimetal Composite Pipe Using Post-Internal-Welding Process[END_REF][START_REF] Liu | Microstructure and corrosion resistance of butt joint of 1Cr18Ni9Ti+ Q235 composite plate[END_REF] and laser welding [START_REF] Missori | Single-pass laser beam welding of clad steel plate[END_REF], are suitable for joining SCSP. However, the differences in chemical compositions and physical properties between the carbon steel and stainless steel make the welding of the SCSP relatively difficult [START_REF] Dhib | Cladding of low-carbon steel to austenitic stainless steel by hot-roll bonding: microstructure and mechanical properties before and after welding[END_REF]. The base material mismatching between the clad layer and the substrate may lead to the generation of a high discontinuous residual stress at the interface between them after welding process [START_REF] Jiang | Numerical simulation to study the effect of repair width on residual stresses of a stainlesssteel clad plate[END_REF]. In addition, Cr and Ni alloy elements in the WM-CL can easily dilute and diffuse into the WM-S, leading to low contents of alloy elements and a low corrosion resistance of the WM-CL as well as a disappearance of the δ-ferrite in the vicinity of the WM-CL interface [START_REF] Ma | Microstructure characterization and properties of carbon steel to stainless steel dissimilar metal joint made by friction welding[END_REF]. Meanwhile, Wang et al. [START_REF] Wang | Welding of duplex stainless steel composite plate: influence on microstructural development[END_REF] reported in their work that a decarburized layer and a carbon-rich layer were also found in the welding interface due to the easy dilution and the carbon diffusion. Furthermore, the excessive diffusion of alloying elements can result in the formation of Cr 23 C 6 carbides at the welding interface, leading to a severe inter-granular depletion (Cr-depletion) and inter-granular cracks [START_REF] An | Microstructure and mechanical properties of stainless-steel clad plate joints produced by TIG and MAG hybrid welding[END_REF].

In case of the SCSP, the preparation of the coupon test prior to welding (i.e., the groove shape) and the selection of the suitable welding sequences are crucial. These preparations may affect the mechanical properties of the SCSP welded joint. Hence, in order to satisfy the requirements in terms of mechanical properties and to ensure a good welding quality, some researchers studied the influence of different groove shapes on the mechanical behaviour and microstructure of the SCSP welded joint [START_REF] Dhib | Cladding of low-carbon steel to austenitic stainless steel by hot-roll bonding: microstructure and mechanical properties before and after welding[END_REF][START_REF] Tian | Welding process of composite plate for construction and its welded microstructure and properties[END_REF][START_REF] Qiu | Analysis on welded joint properties of stainless clad steel plates[END_REF][START_REF] Li | Welding L415/316L Bimetal Composite Pipe Using Post-Internal-Welding Process[END_REF]. While other researchers investigated the effect of the welding sequences on microstructure and mechanical properties of the SCSP welded joint [START_REF] Dhib | Cladding of low-carbon steel to austenitic stainless steel by hot-roll bonding: microstructure and mechanical properties before and after welding[END_REF][START_REF] Tian | Welding process of composite plate for construction and its welded microstructure and properties[END_REF][START_REF] Qiu | Analysis on welded joint properties of stainless clad steel plates[END_REF][START_REF] Li | Welding L415/316L Bimetal Composite Pipe Using Post-Internal-Welding Process[END_REF][START_REF] Liu | Microstructure and corrosion resistance of butt joint of 1Cr18Ni9Ti+ Q235 composite plate[END_REF][START_REF] Missori | Single-pass laser beam welding of clad steel plate[END_REF][START_REF] Jiang | Numerical simulation to study the effect of repair width on residual stresses of a stainlesssteel clad plate[END_REF][START_REF] Ma | Microstructure characterization and properties of carbon steel to stainless steel dissimilar metal joint made by friction welding[END_REF][START_REF] Wang | Welding of duplex stainless steel composite plate: influence on microstructural development[END_REF][START_REF] Yu | Microstructure and mechanical properties of stainless steel clad plate welding joints by different welding processes[END_REF], Li et al. [START_REF] Li | Welding L415/316L Bimetal Composite Pipe Using Post-Internal-Welding Process[END_REF] have studied, firstly, the weld joint performance of the L415/316L cladded steel pipes using four groove shapes. Then, they investigated its microstructures, mechanical properties and corrosion resistances. The results of this paper showed that the U-V shape, with an angle of 60° on the V-shape and with 1 mm as a blunt edge, is the most suitable configuration between the studied four groove shapes. Dhib et al. [START_REF] Dhib | Cladding of low-carbon steel to austenitic stainless steel by hot-roll bonding: microstructure and mechanical properties before and after welding[END_REF] have studied the weld performance of the A283 Gr C/A240 TP316L SCSP using two groove shapes (V-shape and U-V shape). Then, they investigated its mechanical properties. Results showed that failure occurred when they performed the bending test on the V-shape configuration (first configuration). In addition to that, impact toughness of the V-shape configuration was found below the standard requirement (< 27 J). The same authors have also investigated the weld joint performance of the same SCSP (A283 Gr C/A240 TP 316L) using U-V shape and applying two different welding sequences. Results showed that the toughness of the second configuration is below the standard requirement. Moreover, the highest micro-hardness values were observed on the WM-TZ of the second configuration. Results thereby show that proceeding by welding, the parent metal using E7018 followed by welding the transition phase using the ER309L and finishing by welding the clad layer using ER316L (third configuration) hereby improves the welding efficiency of the welded joint. Similar findings were reported by Yu et al. [START_REF] Yu | Microstructure and mechanical properties of stainless steel clad plate welding joints by different welding processes[END_REF] when they investigated the mechanical properties of the SCSP (Q235/SUS304). These authors concluded that welding the clad layer without using the transition phase affects not only the microstructure (excessive diffusion of Ni and Cr) but also the corrosion resistance and mechanical properties. Based on the above discussion, it is clearly seen that the weld joint microstructures and their corresponding mechanical properties are highly dependent on multi-pass welding process parameters and configurations. However, in addition, the microstructure morphology of a welded joint is highly affected by solidification parameters. Thus, it is useful to understand well the relationship that exists between the different weld pool solidification parameters (viz. growth rate R, temperature gradient G, and undercooling ΔT and the resulting microstructure). David et al. [START_REF] David | Correlation between solidification parameters and weld microstructures[END_REF][START_REF] David | Welding: solidification and microstructure[END_REF] have reported that solidification parameters G and R are important in combined forms G×R and G/R since they influence respectively the scale of the solidification morphology and the sub-structure of the weld metal. Trivedi et al. [START_REF] Trivedi | In situ observations of weld pool solidification using transparent metal-analog systems[END_REF] have reported that the solidification front presents a planar character along the fusion line since the ratio G/R is maximal and transforms to cellular and columnar when moving towards the weld centreline (i.e. G/R decreases). Bunaziv et al. [START_REF] Bunaziv | Metallurgical aspects in the welding of clad pipelines-a global outlook[END_REF] have explained that along the fusion line, where the G is largest and the R is smallest, the criterion for plane front stability (G L /R) > (ΔT 0 /D L ), where G L is the thermal gradient in the liquid, D L is the solute diffusion coefficient in the liquid and ΔT 0 is the equilibrium solidification temperature range, is most easily satisfied leading to the presence of a planar structure. They have also explained that throwing the weld centreline, G decreases and R increases promoting therefore a planar instability which gives rise to a cellular and then columnar structure.

In harmony with the above discussed studies and based on the third weld configuration studied by Dhib et al. [START_REF] Dhib | Cladding of low-carbon steel to austenitic stainless steel by hot-roll bonding: microstructure and mechanical properties before and after welding[END_REF], a new hybrid SMAW/GATAW multi-pass welding configuration was proposed and realized in our previous work [START_REF] Ghorbel | Experimental analysis of temperature field and distortions in multi-pass welding of stainless cladded steel[END_REF] to join the SCSP in order to reduce the number of passes and consequently the welding time and cost. The authors have focalized in the first part of their study the effect of the generated heat flux during the multi-pass welding process on the angular distortion and the longitudinal shrinkage of the SCSP welded joint [START_REF] Ghorbel | Experimental analysis of temperature field and distortions in multi-pass welding of stainless cladded steel[END_REF]. Preliminary investigation of the weld mechanical performances including tensile and bend tests showed that the welded joint has higher mechanical tensile strength than the parent metal and that no separations, fractures or tearing appeared on the specimen surface after the bending test. However, the bend specimen showed the presence of a bulging phenomenon located on the welded joint bent surfaces that was explained by the presence of a low hardening zone (LHZ) with low plasticity.

Hence, our previous research [START_REF] Ghorbel | Experimental analysis of temperature field and distortions in multi-pass welding of stainless cladded steel[END_REF] was limited on evaluating the welded joint performances using only conventional tensile and bend tests, which is insufficient to understand the main cause leading to the LHZ apparition. It is useful to further investigate the structural integrity of the welded joint in order to (i) identify the different microstructures within the SCSP-welded joint produced by multi pass SMAW/GTAW process and (ii) understand the relationships that exist between the observed microstructure and the resulting mechanical properties of the welded joint in order to better understand the origin of the LHZ phenomenon.

Materials and methods

Base and fillers materials

Two plates with dimensions of 200 mm × 400 mm were extracted from a cladded sheet metal obtained by a hotroll bonding of a 12-mm low carbon steel (A 283 Gr C) and a 3-mm austenitic stainless steel clad layer (A 240 TP 316L). The dimensional details of the test coupon are given in Fig. 1. The chemical composition and the monotonic mechanical properties of the SCSP base material studied in this work are given respectively in Tables 1 and2.

Welding procedure

In this study, the welding process was carried out using three filler metals E7018, ER316L and ER309L to join the low carbon steel (A283 Gr C), austenitic stainless steel (A 240 TP 316L) and the transition zone, respectively. Typical chemical compositions of these filler metals are given in Table 1. Three passes on the carbon steel side were performed using the SMAW process whereas two passes in a transition phase and four passes on the stainless-steel side using both GTAW process. The used filler metal for each pass and its corresponding welding parameters are given in Table 3. For more details concerning the welding process, see Ref. [START_REF] Ghorbel | Experimental analysis of temperature field and distortions in multi-pass welding of stainless cladded steel[END_REF].

Welded joint characterization

NDE

In order to confirm the welded joint integrity, several nondestructive examination (NDE) tests were performed to detect the presence of somewhat surface and/or volume defects. Indeed, a visual examination, a dye pen examination in the stainless-steel side, a magnetic practical examination in the carbon steel side and a radiographic examination (Fig. 2) were carried out according to ASME code [START_REF]ASME Boiler and Pressure Vessel Code, Section IX: Welding and Brazing Qualifications. An International Code[END_REF] (Section IX QW-302.2).

Micro-and macrostructural analyses

A specimen, with dimensions of 120 × 15 × 9.5 mm, was used to perform micro, macro-graphic and micro-hardness examinations. Both sides of the specimen were mechanically polished using SiC grits papers with a grit number varying from 280 to 1200, followed by a final polishing using 6-, 3-and 1-μm diamond pastes, respectively. The specimen sides were differently etched. The first side was chemically etched, at room temperature, using iron chloride in order to reveal the macrostructure of the welded joint. The second one was prepared in order to examine the welded joint microstructure. For the second specimen side, the carbon steel was chemically etched using 3% of the nitric acid alcohol solution for 5 s while the austenitic stainless steel was electrolytically etched using 10% of the oxalic acid solution in distilled water at 6 V for 20 s. The microstructure of the welded joint was investigated using LEICA® metallographic microscope.

Mechanical characterization setup

In order to characterize the mechanical properties of the joint, several specimens were cut from the welded coupon. Specimens were extracted, from the test coupon using water jet as shown in Fig. 3 and then machined in order to obtain the required dimensions according to the ASME IX code [START_REF]ASME Boiler and Pressure Vessel Code, Section IX: Welding and Brazing Qualifications. An International Code[END_REF]. Tensile and bend tests were carried out, at room temperature, to confirm the reliability of the welded joint as described in ref. [START_REF] Ghorbel | Experimental analysis of temperature field and distortions in multi-pass welding of stainless cladded steel[END_REF]. In addition to tensile and bend tests, toughness and micro-hardness experiments were deeply investigated in this work in order to check the integrity of the joint under dynamic Charpy impact test and correlates mechanical properties to microstructural observations. Indeed, toughness specimens were notched using V-Notch cutter Machine in the WM and the HAZ, respectively, as shown in Fig. 3c. Then, they were placed into a LR-cdw-60® low temperature-testing machine until they reached the temperature of -29 °C. This temperature was fixed since it is corresponds to the operating temperature of containers dedicated to acid conditioning. Charpy tests were performed using JB-W300A® impact testing machine. The absorbed energies were measured, and the fractured surfaces were investigated.

Given the expected complexity of the microstructure in the welded joint, since it presents the assembly of a be-material with three different filler metals, several micro-hardness measurements were carried out on the weld zone, along four different paths, as depicted in Fig. 4. The micro-hardness tests were conducted with Innova® testing machine, using Vickers indenter under a load of 0.1 kg.

Results and discussions

A preliminary study

The welded joint has shown a good visual appearance. Besides, no defects were detected after performing the

(b) (c) (a)
V-Notch in the MW V-Notch in the HAZ Micro-hardness measured lines Fig. 4 Micro-hardness measurement paths on the welded specimen dye pen and magnetic practical examinations. Regarding the radiographic examination, the film revealed a full weld penetration with a complete sidewall fusion as illustrated in Fig. 5. Likewise, the film revealed a welded bead free of any sign of defects. The weld cross section was examined after being etched. Figure 6 reveals the presence of three different zones in the welded joint as follows: the WM-S, the WM-CL and the WM-TZ. In addition to that, the welded joint, shown in this figure, exhibits a full weld penetration with a homogeneous fusion weld in the carbon steel side, stainless steel side and in the transition side. Likewise, the examination of the welded joint and the extent of the HAZ revealed the exemption of any sign of discontinuities or defects, which confirms the obtained NDE results.

Microstructural analysis

In this contribution, a meticulous microstructural investigation of the welded joint was carried out in order to recognise the different phases presented in Fig. 6 as well as their interfaces. The microscopic observations, shown in Figs. 7, 8, 9, 10, 11, 12, 13, 14, revealed the presence of a heterogeneous microstructure resulting from the generation of a chemical and temperature gradient during the welding process [START_REF] Kožuh | Mechanical properties and microstructure of austenitic stainless steel after welding and post-weld heat treatment[END_REF]. In this study, given the complexity of the microstructure that composed the welded zone, the microstructure analyses of the joint are performed in three levels viz: carbon steel side, clad stainless steel side and transition zone.

Carbon steel welded joint side

The microstructure evolution across the welded joint filled with E7018 is shown in Fig. 7. The microstructure of the BM-S is composed of pearlite (dark grains) and ferrite (bright grains) phases (Fig. 7a). In addition, Fig. 7 reveals that pearlitic grains become finer while going from the BM-S zone to the WM-S zone (Fig. 7c) passing through the S-HAZ zone (Fig. 7b). Similar findings were observed by Dhib et al. [START_REF] Dhib | Cladding of low-carbon steel to austenitic stainless steel by hot-roll bonding: microstructure and mechanical properties before and after welding[END_REF] when they studied the metallurgical properties of the SCSP A283 Gr C/A240 TP 316L after welding. In addition, Fig. 7 shows that the microstructure of the carbon steel welded joint side exhibits a typical and healthy structure without any sign of martensitic phase.

Stainless-steel welded joint side

The microstructure of the welded joint zone filled with ER316L is shown in Fig. 8. The examination of the BM-CL reveals the presence of a largely austenitic (γ-white) structure with the presence of some twins (Fig. 8a). Not far from the BM-CL, the microstructure observed along the fusion line shows that the crystallization occurred according to an epitaxial mode (Fig. 8b). Indeed, the solidification growth occurs by arranging atoms in the liquid phase on the existing BM-CL crystalline, thus extending it without altering the original crystallographic orientation. As a result, grain boundaries from the BM-CL are continuous across the fusion line, forming solidification grain boundaries in the WM-CL [START_REF] An | Microstructure and mechanical properties of stainless-steel clad plate joints produced by TIG and MAG hybrid welding[END_REF][START_REF] Nelson | Investigation of boundaries and structures in dissimilar metal welds[END_REF][START_REF] Ming | Microstructural characterization of an SA508-309L/308L-316L domestic dissimilar metal welded safe-end joint[END_REF]. Far away from the fusion line, the microstructure of the WM-CL (Fig. 8c) presents an austenite matrix with a dendritic δ-ferrite, which mainly solidified in skeletal and lathy morphologies. This type of microstructure depends on the WM-CL solidification mode. The skeletal and lathy morphology of the δ-ferrite theoretically depends on the solidification rate of the WM-CL, which is mainly controlled by the diffusion of the Cr and Ni [START_REF] Feng | Keyhole gas tungsten arc welding of AISI 316L stainless steel[END_REF]. Indeed, and In order to calculate the percentage of [Cr] eq and [Ni] eq in WM-CL zone, a spark optical emission spectroscopy (OES) technique was used to determine the chemical composition of this zone. The found result is summarised in Table 4.

According to Fig. 9, the solidification mode of the WM-CL zone is a primary fer ritic solidification, with ferrite to austenite formation (FA mode). Chassignole [START_REF] Chassignole | Influence de la structure métallurgique des soudures en acier inoxydable austénitique sur le contrôle non destructif par ultrasons[END_REF] reported that the δ-ferrite, in the FA solidification mode, crystallizes at first. Then, the δ -> γ transformation occurs during the cooling process. Since this transformation is incomplete, [START_REF] Kaya | An investigation into the explosive welding/cladding of Grade A ship steel/AISI 316L austenitic stainless steel[END_REF] [Cr] eq = %Cr + %Mo + 1.5%Si + 0.5%Nb [START_REF] Rao | Weld overlay cladding of high strength low alloy steel with austenitic stainless steel -structure and properties[END_REF] [Ni] eq = %Ni + 30%C + 0.5%Mn the residual δ-ferrite remains in the WM-CL after solidification.

Idem with the observations shown on the carbon steel welded joint side, the microstructure of the stainless steel welded joint side reveals a healthy structure without any sign of martensitic phase.

Transition phase welded joint

In this section, the microstructure evolution of the welded joint zone filled with ER309L is investigated (Fig. 10). Figure 10a reveals that the S-HAZ microstructure is almost pearlitic with a predominant appearance of ferrite coarse grains located at the S-HAZ adjacent to the WM-TZ. The disappearance of the pearlitic grains may be ascribed to the carbon depletion, which occurs during the welding process. Li et al. [START_REF] Li C'a, Qin | Microstructures and mechanical properties of stainless-steel clad plate joint with diverse filler metals[END_REF] reported in their study that due to the element diffusion, a decarburized layer is formed in the S-HAZ and the precipitated carbon is aggregated along the fusion line. The appearance of the ferrite coarse grains may be attributed to the grain coarsening behaviour under the effect of high temperature generated during the welding process, as reported by An et al. [START_REF] An | Microstructure and mechanical properties of stainless-steel clad plate joints produced by TIG and MAG hybrid welding[END_REF]. In analogy with the bonding phenomenon that occurs, at high temperature, between the clad layer and the substrate, Dhib et al. [START_REF] Dhib | Mechanical bonding properties and interfacial morphologies of austenitic stainless steel clad plates[END_REF] reported in their work that carbon element can easily migrate from BM-S forwards BM-CL due to the high mobility of its small atomic radius (0.077 nm). The same authors have also reported that grain coarsening and decarburization have occurred at the BM-S near the interface due to long exposure time at high temperature.

The same phenomena were also observed in Fig. 10b while examining the WM-S/WM-TZ interface. The layer thickness of coarse grains located at WM-S/WM-TZ interface exhibits an average value of 55 ± 7 μm, which is smaller than that located at the S-HAZ/WM-TZ interface (average of 75 ± 6 μm) (Fig. 10a). The difference in the layer thickness values can be explained by the difference in carbon percentage between A283 Gr C (C (wt %) = 0.167) and E7018 (C (wt %) = 0.23). Indeed, during the welding, a decarburization of the S-HAZ and the WM-S occurs until the WM-TZ reaches its nominal carbon content. Since E7018 has high carbon content, the carbon depletion in the WM-S near the interface will occur with a small width of the decarburized 10a and b also reveal the presence of a dark layer 25 ± 3 μm wide. Due to the excessive diffusion of the carbon element from BM-S and WM-S zones to WM-TZ, a carbon-rich layer with a dark aspect called carburized layer is observed [START_REF] An | Microstructure and mechanical properties of stainless-steel clad plate joints produced by TIG and MAG hybrid welding[END_REF]. The comparison between thickness values of carburized and decarburized layers shows that the decarburized layer is always thicker than the carburized layer. This result may be attributed to the difference of diffusion velocities and the carbon element solubility in austenite and ferrite phases [START_REF] An | Microstructure and mechanical properties of stainless-steel clad plate joints produced by TIG and MAG hybrid welding[END_REF]. Actually, the carbon diffusion can be easily pronounced thanks to its small atomic radius (0.077 nm), which offers it great mobility. In addition, the microstructure of the WM-TZ shown in Fig. 10c revealed the presence of martensitic laths and cellular and columnar sub-grains elongated according to their local solidification direction.

The microstructure of the WM-TZ has undergone several complex thermophysical phenomena, during the multi-pass welding process, which are related to the re-melting and solidification of the welded pool. The grain morphology transformations are due to the combined effects of both temperature gradients (G) and the rate of grain's growth (R) generated during the solidification process. The of the temperature gradient and the growth rate R on the morphology and the size of the solidified microstructure is represented in Fig. 11. When carrying out the subsequent pass with ER309L, a molten pool will be formed and then solidified. Since the ratio G/R is maximal at the bottom of the molten pool (i.e., near the fusion boundary), there will be few planar crystals that firstly form. With the movement of the solid/liquid interface towards the weld pool centreline, the solidified microstructure progressively changes from planar crystals to cellular and then to columnar due to the decrease of the ratio G/R. The same result was confirmed by Li et al. [START_REF] Li | Microstructure evolution and mechanical properties of multiple-layer laser cladding coating of 308L stainless steel[END_REF] when they studied the microstructure and mechanical property evolution of multipass laser welding. These authors have also reported that when the bottom of the molten pool is being solidified, the top of this molten pool is also being solidified into dendrite direction because of the change in G direction under the effect of the natural convection. However, since the solidification rate from the bottom to surface is much faster than that from surface to bottom, the columnar d accounts for a large area in the welded bead. The same authors [START_REF] Li | Microstructure evolution and mechanical properties of multiple-layer laser cladding coating of 308L stainless steel[END_REF] have also reported that the effect of the multi-pass welding has not only affected the microstructure but also the mechanical properties of the welded joint. Rathod et al. [START_REF] Rathod | Diffusion control and metallurgical behavior of successive buttering on sa508 steel using Ni-Fe alloy and inconel 182[END_REF] reported in their work that the cellular and columnar structures are characterized by their martensitic nature that can affect the mechanical properties of the weld.

In order to confirm the observed microstructure, it is useful to predict which phase is present in the WM-TZ zone using Schaeffler diagram as previously explained. However, in our case study, OES analysis cannot be performed to determine the chemical composition the WM-TZ zone given its small cross-section area compared to dimensions of sparking area. For this, another technique was used to predict the presence of the martensitic structure. [Cr] eq and [Ni] eq of respectively A 283 Gr C and ER309L were calculated (Table 4) and plotted on the Schaeffler diagram, as shown in Fig. 12. According to this diagram, the martensiteaustenite mixed zone can be formed in regions where the dilution ratio is between 31 and 53%. For this, the dilution was then calculated basing on the macro-graphic cross section shown in Fig. 6. The dilution can be defined as the contribution of the BM-S into the WM-TZ (Fig. 13). The dilution was calculated using Eq. 3, the dilution's value was found 32%, and the presence of a martensitic structure is confirmed (Fig. 12).

The fusion boundary (FB) in the vicinity of the carburized layer (i.e. between the WM-TZ and the BM-S) was evaluated. A type-II boundary running parallel to the FB is found in the WM-TZ, as shown in Fig. 14. This type-II boundary parallels the fusion boundary approximately 40 μm away from the WM-TZ. Type II boundaries may be a result from a change in the primary mode of solidification. Actually, the primary solidification occurs as a ferrite (bcc) in the S-HAZ and as an austenite in the WM-TZ. At high temperature, the S-HAZ will undergo an allotropic δ-γ transformation. The fusion line becomes, therefore, an austenite (fcc) boundary between the S-HAZ (fcc) and the WM-TZ (also fcc) in the austenitic temperature range. Since both sides have an austenitic lattice, the austenitic boundary can migrate easily into the WM-TZ. This migration is principally the result of the composition gradients within the transition region between the S-HAZ and the WM-TZ. [START_REF] Wu | Formation of crack-susceptible structures of weld overlay of corrosion resistant alloys[END_REF][START_REF] Nelson | Nature and evolution of the fusion boundary in ferritic-austenitic dissimilar weld metals, Part 1-Nucleation and growth[END_REF].

Several researchers prove the formation of a martensitic structure in the type-II boundary. Rowe et al. [START_REF] Rowe | Hydrogen-induced cracwking along the fusion boundary of dissimilar metal welds[END_REF] reported in their study that the intermediate composition along the drawn line on the Schaeffler diagram (i.e. between ER309L and A 283 Gr C) would develop martensitic and austenitic plus martensitic microstructures. Nelson et al. [START_REF] Nelson | Nature and evolution of the fusion boundary in ferritic-austenitic dissimilar metal weldspart 2: on-cooling transformations[END_REF] prove that if the dilution is between 20 and 80%, the microstructure of the WM-TZ will be observed as a mostly austenitic microstructure with a band of martensite along the FB. Based on the above discussion, we can conclude that the microstructure of the type-II boundaries is a martensitic structure since the dilution is 32%. Furthermore, type-I boundaries, running roughly perpendicular to the FB between the equiaxed grains as a result of an epitaxial growth, have also been found [START_REF] Rowe | Hydrogen-induced cracwking along the fusion boundary of dissimilar metal welds[END_REF].

Mechanical characterization

Micro-hardness

The micro-hardness measurements conducted along four paths on the welded specimen (Fig. 4) are depicted in Fig. 15. The micro-hardness profile performed along L1 direction (Fig. 15a) shows that the hardness values measured on the S-HAZ surface (average 208 ± 8 HV) are slightly higher than those measured on the WM-S (average 213 ± 5 HV) and the BM-S (average 171 ± 9 HV). The highest micro-hardness level measured on the S-HAZ can be ascribed to the perlite dissociation, which is observed in Fig. 7b. In contrast, the micro-hardness values measured on the WM-S are lower, but still higher than those measured on the BM-S owing to the high cooling rate and grain refinement at the WM-S (Fig. 7c).

Figure 15b displays the micro-hardness profile of the welded joint along L2 direction. It is obvious that microhardness values measured on the S-HAZ near the interface (average 429 ± 30 HV) are significantly higher than those measured on the WM-TZ (average 384 ± 14 HV) and the BM-S (average 171 ± 17 HV). The highest micro-hardness level measured on the S-HAZ can be assigned to the carburized layer shown in Fig. 10a along the FB. Herein, it is obviously clear from Figure 15b that the highest microhardness values are in the vicinity of the fusion line. Indeed, the carbide forms in this area due to the chrome and carbon diffusion phenomenon at high temperature leading to the formation of the type I and type II boundaries as clearly seen in Fig. 14. However, the micro-hardness values measured on the WM-TZ are lower, but still higher than those measured on the BM-S. This can be ascribed to the presence of the martensite laths as already observed in Figure 10c.

Figure 15c shows the micro-hardness curve across the L3. The micro-hardness values measured on the WM-CL vary slightly (average 221 ± 19 HV) and are found to be The micro-hardness profile along L4 direction is shown in Fig. 15d. It is clear that both of WM-CL and WM-S have shown no large variation in micro-hardness values. In contrast, the micro-hardness value of the WM-TZ increased significantly and reached about 406 HV. This can be attributed to the presence of the martensitic phase previously observed in Fig. 10c as well as the presence of the carbide agglomerated in the FB and observed as type I and type II boundaries (Fig. 14).

On the bases of the previous discussion, the hardening zone shown in the WM-TZ and its S-HAZ are confirmed not only by the presence of the martensitic phase but also by the carburation/decarburization phenomena, which accelerate the formation of the chromium carbide leading thereafter to a hardening at the WM-TZ/S-HAZ interface. This hardening zone may adversely affect the bend test and impact toughness properties of the welded joint due to the inhomogeneity.

Tensile and bend test

The transverse tensile strength specimens have been evaluated in our previous investigation [START_REF] Ghorbel | Experimental analysis of temperature field and distortions in multi-pass welding of stainless cladded steel[END_REF]. The main results are summarized in Table 5.

It has been reported in the work of Ghorbel et al. [START_REF] Ghorbel | Experimental analysis of temperature field and distortions in multi-pass welding of stainless cladded steel[END_REF] that one of the significant outcomes of these tensile tests is that the two specimens were fractured out of the welded joint area (i.e. failure is localized in the BM). This result shows that the weld zone is more resistant than the base material, which complies with the ASME IX requirement.

In addition, bending tests, applied on the four side bend specimens [START_REF] Ghorbel | Experimental analysis of temperature field and distortions in multi-pass welding of stainless cladded steel[END_REF], indicated that no separations, fractures or tearing was observed on all bent surfaces which is in accordance with the industrial code ASME IX [START_REF]ASME Boiler and Pressure Vessel Code, Section IX: Welding and Brazing Qualifications. An International Code[END_REF]. However, the bulging phenomenon, which appeared in the weld area after bending, was previously explained by the potential presence of a LHZ with low plasticity in the WM-TZ. Based on the previous results, the main cause producing the LHZ in the WM-TZ is the presence of a martensitic structure in which was observed microscopically and recognized by its high micro-hardness values.

Charpy V-notched study

Toughness tests were performed on the Charpy V-Notch specimens as shown in Fig. 3c. The main values of the WM and the HAZ toughness are plotted in Fig. 16a and b respectively. It is clearly seen that the WM toughness showed the highest values with an average of 80.2 J. However, the average of the HAZ toughness values (22.9 J) was slightly lower than the standard requirement (27 J). Herein, in order to get a coherent understanding of the obtained values, the fractured surfaces of both WM and HAZ specimens were evaluated. Figure 17 demonstrates the fractured surfaces of the Charpy impact specimens. The WM (Fig. 17a) showed two types of fracture appearances. Dimples, micro-voids and shear lips, which were induced by the shearing stress in the sliding plane, appear on the bottom of the fractured surface, while mixture of fibrous and radial zone appears on the top of the fractured surface. A small shear lip in this region can be recognised. The average value of the WM lateral expansion observed in Fig. 16a was found about 1.32 mm. It can be noted that fracture occurred by a ductile mode on the bottom and a mixed ductile and cleavage mode on the top. These findings hereby confirm the amount of energy absorbed on the WM specimens (80.2 J).

On the other hand, the optical fracture surface of the HAZ (Fig. 17b) can be divided into four parts: prefabricated V-Notch, fibrous region, radial region and shear lip. Each part is corresponding to different fracture features. The fracture initiated from the fibrous region adjacent to the prefabricated V-Notch. The surface is featured with fibrous pattern. The radial region, which located in the middle of the fractured surface, was featured with a brittle cleavage with bright and reflective facets. A small shear lip was observed as a final rupture induced by the shearing stress in the sliding plane. The average value of the HAZ lateral expansion observed in Fig. 16b was found about 0.46 mm. It can be noted that fracture occurred by a mixture of ductile-brittle mode with predominated brittle fracture appearance. These findings hereby confirm the amount of energy absorbed on the HAZ specimens (22.9 J). It is reasonable to link the mode of the fracture toughness with the variation of microstructure and micro-hardness. It can be noted that the ductility in the HAZ decreases due to the coarse grains observed in Fig. 10, the formation of martensitic structure and the high micro-hardness observed in Fig. 15.

Conclusion

In this manuscript, the structural integrity of the studied SCSP welded joint, produced by a hybrid SMAW/GTAW multi-pass welding process, was investigated through nondestructive examinations, microstructural observations and mechanical characterisation including tensile, bend, microhardness and impact toughness tests. As a result of this study, the following conclusions can be drawn:

• The microscopic examination carried out on the welded joint showed a typical ferritic-pearlitic structure and austenitic matrix with a dendritic δ-ferrite, which solidified in skeletal and lathy morphologies, respectively in the carbon steel and in the stainless steel weld sides. No sign of martensite was found in these investigated weld regions. • The microscopic examination performed on the WM-TZ showed the presence of martensite laths and cellular and columnar structure characterized by their martensitic nature. It was revealed in the vicinity of the fusion boundary (FB) the presence of a decarburized layer generated by the carbon depletion from the substrate heataffected zone (S-HAZ) during the welding process and its agglomeration along the fusion line. Despite the selection of the filler metal ER309L in the studied process, to avoid the formation of the martensite near the BM-S/WM-TZ interface, thanks to its Ni content that acts as a diffusion barrier, type I and II boundary joints, which are characterized by their martensitic nature, were appeared.

• The micro-hardness tests showed that the highest microhardness values were recorded in the WM-TZ and in vicinity of the FBs with an average of 429 ± 30 HV, which confirms the presence of a martensitic structure. • Charpy impact test results showed that the average value of the WM and the HAZ fracture toughness was 80.2J and 22.9J, respectively. The relatively low fracture energy of the HAZ was highlighted by the brittle feature of the fractured surface. • In view of the reached outcomes, it can be retained that the main cause producing the LHZ is the martensitic structure observed in the WM-TZ along the fusion line. Martensitic structure can be avoided by adjusting the heat input parameters in such a way that the percentage of the dilution in the WM-TZ does not exceed 31% in accordance with Schaeffler diagram. The effect of the subsequent passes on the previous welded beads will be the subject of our future work.

Fig. 1

 1 Fig.1Form and dimensions of the prepared plates and the butt-weld U-V shape for welding process

Fig. 2

 2 Fig. 2 Setting up the radiographic test coupon; a the film cassette on stainless steel side and b the IQI, ruler and writing on the carbon steel side

Fig. 3

 3 Fig. 3 Form and dimensions (in mm) of specimens extracted from the welded plates. a Tensile test specimen. b Bend test specimen. c Impact test specimen

Fig. 5 Fig. 6 Fig. 7 Fig. 8

 5678 Fig. 5 Radiographic film of the welded joint

Fig. 9

 9 Fig.9Solidification modes of austenitic stainless steels based on different Cr eq /Ni eq values[START_REF] Feng | Keyhole gas tungsten arc welding of AISI 316L stainless steel[END_REF]

Fig. 10

 10 Fig. 10 Metallographic examination of the weld metal transition zone. (a) Weld Metal Transition Zone/ Base Metal Substrate interface (b) Weld Metal Transition Zone/ Weld Metal Substrate interface (c) Weld Metal Transition Zone

Fig. 11 Fig. 12

 1112 Fig.11Effect of temperature gradient G and growth rate R on the morphology and size of solidification microstructure[START_REF] Kou | Welding metallurgy[END_REF]

Fig. 13 Fig. 14

 1314 Fig.[START_REF] Li | Welding L415/316L Bimetal Composite Pipe Using Post-Internal-Welding Process[END_REF] Schematic view showing the calculation of dilution in a fusion weld

Fig. 15

 15 Fig. 15 Micro-hardness of the welded joint along: a L1, b L2, c L3 and d L4 profiles

Fig. 16

 16 Fig. 16 Measured Charpy V-notch energy and lateral expansion on a weld metal specimens and b heat-affected zone specimens

  Toughness H eat Affected Zone Lateral expansionLateral Expansion (mm) Energy (J)

Fig. 17

 17 Fig.[START_REF] Ma | Microstructure characterization and properties of carbon steel to stainless steel dissimilar metal joint made by friction welding[END_REF] The fractured surface observed on the impact test specimen of a WM and b HAZ

Table 1

 1 Chemical composition of the SCSP and filler metals (wt. %)

		Elements	C	Si	Mn	P	S	Cr	Ni	Mo
	Base material A283 Gr C	0.167 0.172 0.479 0.019 0.022 -	-	-
		A240 TP 316 0.017 0.514 1.124 0.038 0.002 16.654 10.109 2.092
	Filler metals	ER 316L	0.08	0.53	1.17	0.053 0.008 16.18	10.55	1.90
		E7018	0.23	0.171 0.402 0.032 0.012 0.024	0.021	< 0.002
		ER 309L	0.025 0.45	1.8	0.02	0.02	24	13	-

Table 3

 3 Multi-pass welding process parameters used for joining the studied material DCRP direct current reverse polarity, DCSP direct current straight polarity

	Weld	Filler metal Filler	Current and polarity Current (A) Voltage (V) Welding	Heat
	pass		metal Ø				speed	input (kJ/
	No.		(mm)				(mm/s)	mm)
	1	E7018	2.5	DCRP*	85	22	1.43	1.31
	2	E7018	3.2	DCRP	116	22	1.13	2.27
	3	E7018	3.2	DCRP	113	24	1.86	1.46
	4	ER309 L	2.4	DCSP**	133	10	1.3	1.02
	5	ER309 L	2.4	DCSP	141	11	1.18	1.31
	6	ER316 L	2.4	DCSP	142	11	1.1	1.42
	7	ER316 L	2.4	DCSP	140	11	0.93	1.66
	8	ER316 L	2.4	DCSP	130	10	1.25	1.04
	9	ER316 L	2.4	DCSP	133	10	0.86	1.55

Table 4

 4

	Chemical composition of the WM-CL (wt. %) and	Element	C	Si	Mn	Cr	Ni	Mo	Nb	[Cr] eq	[Ni] eq	[Cr] eq /[Ni] eq
	[Cr] eq /[Ni] eq ratio	WM-CL	0.053 0.333 2.41	19.48 11.43 1.61 0.057 21.637 14.225 1.52
		(analysed
		zone)
		BM-S	0.167 0.172 0.479 -	-	-	-	0.26	5.25	-
		ER309L	0.025 0.45	1.8	24	13	-	-	24.68	14.65	-

Table 5

 5 Monotonic tensile properties of the welded clad material at room temperature[START_REF] Ghorbel | Experimental analysis of temperature field and distortions in multi-pass welding of stainless cladded steel[END_REF]

	Tensile strength Rm (MPa)	487 ± 2.8
	Yield Strength Re (%)	423 ± 2.5
	Elongation A (%)	31.4 ± 1.4

MSMP-EA7350, Arts et Métiers ParisTech, 2 cours des Arts et Métiers, 13617 Aix-en-Provence, France

Acknowledgements

The authors would like to thank SOCOMENIN Company for their support. They equally would like to thank Mr M.T. Hbaieb and A. Badri, staff engineers at SOCOMENIN Company, for their knowledgeable help and support.

Declarations

Abbreviations

Conflict of interest

The authors declare no competing interests.

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