Mercedes Bonet
email: mercedes.bonet-semenas@inserm.fr

PhD Laetitia Marchand

MSc Monique Kaminski

MSc Anne Fohran

Aisha Betoko

Marie-Aline A Charles

MD MPH Béatrice Blondel

Aishabetoko B Mph

Marie-Aline Mph

Charles

M De Agostini

A Forhan

B Heude

M J Saurel-Cubizolles

P Dargent

X Fritel

B Larroque

N Lelong

C Nabet

V Goua

G Magnin

R Hankard

Breastfeeding duration, social and occupational characteristics of mothers in the French 'EDEN mother-child' cohort

Keywords: breastfeeding duration, employment, socioeconomic status, France Background

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

Exclusive or full breastfeeding rates at 3-4 months varied widely in Europe in 1998-2002, from 70-60% in Scandinavian countries to 35% in the Netherlands [START_REF] Cattaneo | Protection, promotion and support of breast-feeding in Europe: current situation[END_REF] and 16% (at 3-5 months) in the UK [START_REF] Hamlyn | Infant Feeding 2000[END_REF], and any breastfeeding rates ranged from 90-75%, to47%(1) and 30%(2) respectively. In France, no reliable national data on breastfeeding duration is available but several studies suggest that breastfeeding rates are low.(3) Any breastfeeding rates of 15% at 3-4 months (in 2000) have been reported in the literature [START_REF] Cattaneo | Protection, promotion and support of breast-feeding in Europe: current situation[END_REF] and studies based on small samples in the 1990"s suggested a median duration of any breastfeeding of around 10 weeks. [START_REF] Castetbon | Drawlines for the surveillance of breastfeeding in France[END_REF] Any breastfeeding rates at the maternity unit were also lower in France than in other European countries [START_REF] Cattaneo | Protection, promotion and support of breast-feeding in Europe: current situation[END_REF] in the early 2000"s, though they had significantly increased from 53% in 1998 to 62% in 2003 and 69% in 2010 [START_REF] Blondel | Enquête Nationale Perinatale 2010: les naissances en 2010 et leur évolution depuis 2003[END_REF].

Socio-demographic characteristics of mothers have been associated with exclusive breastfeeding duration in Scandinavian [START_REF] Lande | Infant feeding practices and associated factors in the first six months of life: the Norwegian infant nutrition survey[END_REF][START_REF] Ludvigsson | Socio-economic determinants, maternal smoking and coffee consumption, and exclusive breastfeeding in 10205 children[END_REF] and other European [START_REF] Lanting | Infant milk feeding practices in the Netherlands and associated factors[END_REF][START_REF] Skafida | Juggling Work and Motherhood: The Impact of Employment and Maternity Leave on Breastfeeding Duration: A Survival Analysis on Growing Up in Scotland Data[END_REF] countries. These studies have shown that mothers who are older, married, multiparae, from a high social class or non-smokers breastfeed longer. Maternal occupational characteristics might also affect breastfeeding duration. In the early 2000"s, studies in the UK [START_REF] Bolling | The Information Centre Part of the Government Statistical Service[END_REF] [START_REF] Hawkins | The impact of maternal employment on breast-feeding duration in the UK Millennium Cohort Study[END_REF] [START_REF] Skafida | Juggling Work and Motherhood: The Impact of Employment and Maternity Leave on Breastfeeding Duration: A Survival Analysis on Growing Up in Scotland Data[END_REF] and the Netherlands [START_REF] Lanting | Infant milk feeding practices in the Netherlands and associated factors[END_REF] showed that maternal employment, specially full-time employment, was negatively associated with breastfeeding. Little is known about the independent effect of maternal occupational characteristics, particularly the time of maternal return to work and the full/part-time employment status, on exclusive breastfeeding duration.

Differences in parenthood and labour policies in European countries might have an impact on both breastfeeding practices and maternal employment [START_REF] Galtry | The impact on breastfeeding of labour market policy and practice in Ireland, Sweden, and the USA[END_REF]. France, the UK and the Netherlands hada relatively high percentage of working mothers (not including mothers on maternity or parental leave) with children less than 3 years of age: 47%, 52% and 65% respectively in 2006 [START_REF]OECD Family database[END_REF]. However, maternity leave systems differ across these countries in their eligibility criteria for maternity leave payments and maximum duration. For instance, maternity leave maximum duration is 16 full paid weeks (up to 2773 euros per month in 2006) for the 1 st and for the 2 nd child and 26 weeks for subsequent children in France; 16 full paid weeks in the Netherlands; and 26 paid weeks in the UK (90% of average weekly earnings for the first 6 weeks and a statutory maximum of GBP108.85 per week (in 2006) for the following weeks) followed by 26 unpaid weeks. Some European countries also offer the possibility to prolong the leave period [START_REF]OECD Family database[END_REF], such as France where mothers also have the opportunity to take a parental leave for 3 years or to reduce the number of working hours.

The French situation concerning breastfeeding and maternal work was particularly interesting in the mid-2000"s, as breastfeeding rate was relatively low(1) and maternal employment high [START_REF]OECD Family database[END_REF]. Our aim was to study the associations between any and almost exclusive breastfeeding practices at 4 months of infant"s age and socio-demographic and occupational characteristics of the mothers (time of maternal return to work and full-time employment), in a French mother-child cohort.

Methods

Data collection

The EDEN (Etude des Déterminants pré et postnatals précoces du développement et de la santé de l"ENfant) is a prospective mother-child cohort in two French university hospitals.Its primary aim is the study of prenatal and early postnatal nutritional, environmental, and social determinants of children"s development and health [START_REF] Drouillet | Maternal fatty acid intake and fetal growth: evidence for an association in overweight women. The 'EDEN mother-child' cohort (study of pre-and early postnatal determinants of the child's development and health)[END_REF]. This cohort was not specifically designed to study breastfeeding practices. Pregnant women were recruited before [START_REF] Baker | Maternal employment, breastfeeding, and health: evidence from maternity leave mandates[END_REF] Freedom" (CNIL). Written consent was obtained from the mother for herself at inclusion and for her newborn child after delivery.

An interview was carried out by a midwife during pregnancy to collect data on maternal characteristics: age, parity, educational level, family income, family origin, partnership status (marital status and living with a partner),tobacco consumption, employment (yes/no) and fulltime or part-time employment during pregnancy. Medical records were used to extract data on mode of delivery, infant"s characteristics at birth and during hospital stay (gestational age, birth weight and sex) and mode of infant feeding at discharge. Parents answered postal questionnaires at 4, 8 and 12 calendar months to collect data on infant feeding, partnership status, tobacco consumption, date of return to work after birth, and full-time or part-time employment.

Partnership status at 4 months after birth related to marital status (married or not) and whether the woman was living with a partner. Educational level referred to the highest diploma obtained: less than high-school, high school diploma, some university (at least a 2year university degree) and university degree (at least a 3-year university degree). Family origin distinguished two groups: mother, father or a grand-parent born in France (French), or one of these family members born abroad (foreign). Smoking at 4 months after the birth was classified as follows: non-smoker before pregnancy, smoker before pregnancy but nonsmoker at 4 months, and smoker before pregnancy and at 4 months after birth. Return to work after birth referred to time of return to work (at 4 months after birth or before, between 5 and 8 months after birth, after 8 months). Full-time or part-time employment was known during pregnancy and at 12 months.

In each postal questionnaire, mothers described the infant"s consumption of breast milk, formula, cow"s milk, bottled water (up to 3 completed months of age), tap water, other fluids, and solids. At 4 months, infant feeding information was collected for the first week, the 2 nd to 4 th weeks of life and then for each month between the 1 st and 3 rd months of infant age. At 8 and 12 months, mothers retrospectively answered the question: "How old was your baby when you introduced the following foods regularly: formula, cow"s milk, tap water and solids?". In the three questionnaires, mothers answered the question: "Do you still breastfeed your infant?" and if they answered "No", they were asked to give the date when they had stopped.

Almost exclusive breast infants received breast milk as their only source of milk, but they could have received other liquids or food. Any breastfeeding included all infants receiving breast milk: almost exclusive breastfeeding infants and those receiving partial breastfeeding (breast milk and formula).

Statistical analysis

All mothers (n=1339) breastfeeding at discharge from the maternity unit (71% of the 1889 women with feeding status information at discharge) were selected. Data on infant feeding at 4 months of infant"s age was available for 92% (n=1233) of the infants receiving any breastfeeding at discharge. We considered any and almost exclusive breastfeeding at discharge as breastfeeding during the first week of life, because the average length of stay in maternity unit was 5.1 days in Nancy and 4.4 days in Poitiers. We then calculated breastfeeding rates at the 3 rd completed week of life, and then at each completed month from 1 to 12 months of the infant"s age.

Factors associated with breastfeeding at 4 completed months of infant"s age (5 th month of life) were studied. We chose this age because about half of breastfeeding mothers (44%) had returned to work then: 15% before 4 months after the birth and 29% during the 4 th month. In addition, changes in the French recommendations on breastfeeding duration, from 4 to 6 months( 14), were quite recent when we started recruiting the cohort.

The influence of the time of return to work and full-or part-time employment status at 12 months were studied, in the sub-sample of mothers who worked during pregnancy (n=979).

As full-or part-time employment status was unknown at the time of return to work, we also studied the association between breastfeeding and employment status during pregnancy.

Maternal occupation was strongly correlated with time of return to work and full or part-time employment, and therefore was not included in our analysis. Mothers with professional or intermediate occupations tended to return to work before 4 months and were more likely to work full-time at 12 months. In contrast, manual workers and shopkeepers tended to return to work later and to work part-time.

Pearson"s  2 tests were used for bivariable analyses. Logistic regression models were used to study the associations between breastfeeding at 4 months of infant"s age and variables significantly associated, at p<0.20, with breastfeeding in bivariable analysis. Statistical analysis was performed using SAS 9.2 (SAS Institute Inc., Cary, NC, USA).

Results

At discharge, 91.9% (n=1230) of infants included in our analysis were almost exclusive breastfed and 8.1% (n=109) were receiving mixed feeding (breast milk and formula).

Any breastfeeding decreased slowly between discharge and the 3 rd completed week after birth (Figure 1), and continued to decrease steadily between the 1 st and the 3 rd months of infant"s age. Decreases were more marked for almost exclusive breastfeeding: the proportion of infants almost exclusive breastfed decreased by 17% in the first weeks after discharge and by around 20% each month between the 1 st and 3 rd completed months. At 4 months of infant"s age 42% of the mothers breastfeeding at discharge were giving any breastfeeding and 20% almost exclusive breastfeeding.

Breastfeeding rates at 4 months of infant"s age, both any and almost exclusive breastfeeding rates, were higher among older women, multiparae, those with a higher educational level and those with a foreign family origin (Table 1). Smoking women were less likely to breastfed than women who did not smoke at 4 months. Rates of any breastfeeding were high among both low-and high-income women, and those who did not return to work before 4 months.

Almost exclusive breastfeeding was high among low-income women, but very low among women who had returned to work before 4 months. Girls were breastfed longer than boys.

Breastfeeding at 4 months was not statistically associated with mode of delivery, gestational age, birthweight, or study centre.

After adjustment for all factors in Table 1, women were more likely to continue any or almost exclusive breastfeeding if they had a universitydegree, foreign family origin, a low familyincome, had not returned to work at or before 4 months after birth and did not smoke before pregnancy. Girls were breastfed for longer than boys.

Table 2 shows breastfeeding rates at 4 months for women who worked during pregnancy. Any breastfeeding at 4 months of infant"s age was higher among women returning to work after 4 months. Almost exclusive breastfeeding at 4 months increased significantly with a later return to work. Rates did not differ between women working full-time or part-time after birth. Few mothers returning to work in the first year after delivery combine breastfeeding and work: 58% had stopped breastfeeding before returning to work and 11% stopped during the month they returned to work.

After adjustment, women were more likely to continue breastfeeding if they returned to work later, had a high level of education or had a low family-income. There were no statistical differences according to full-or part-time employment at 12 months or during pregnancy (data not shown for the employment status during pregnancy). No statistical differences were found when mothers working part-time during pregnancy and at 12 months were compared to mothers working full-time. Figure 2 shows a rapid decrease in any breastfeeding in the first months after birth for women who returned to work earlier (at 4 months after birth or before), but a slower decrease for women returning to work more than 4 months after birth.

Discussion

Any breastfeeding decreased slowly in the first weeks after discharge from the maternity unit, and continued to decrease steadily between the 1 st and 3 rd completed months of infant"s age.

Decreases were more marked for almost exclusive breastfeeding compared to any breastfeeding, particularly between discharge and the 3 rd completed week after birth. Women were more likely to breastfeed, both any or almost exclusive breastfeeding, at 4 months if they had a universitydegree, a low family-income or did not smoke. Women who returned to work before 4 months of infant"s age were less likely to continue to breastfeed than women who returned to work later, independently of being full-or part-time workers. The association was stronger for almost exclusive breastfeeding mothers than for any breastfeeding ones. The EDEN cohort is not representative of the French population of newborn infants.

Compared to the national sample of births in the National Perinatal Surveys of 2003 and 2010 [START_REF] Blondel | Enquête Nationale Perinatale 2010: les naissances en 2010 et leur évolution depuis 2003[END_REF], women in the present cohort were more educated, but it is difficult to know how the higher social level of the cohort could have affected our results. Nevertheless the associations between maternal socio-demographic characteristics and breastfeeding at the maternity unit in this cohort (data available at request) were similar to those observed at national level [START_REF] Bonet | Differential trends in breastfeeding according to maternal and hospital characteristics: results from the French National Perinatal Surveys[END_REF]. Using the EDEN cohort we obtained high quality data on infant feeding.

Data were verified by cross-checking infant feeding information obtained at four different points during the first year of life; missing values at 4 months were completed using subsequent questionnaires; recall periods of infant feeding were short as mother reported infant feeding practices every 4 months, and provide valid and reliable estimate of breastfeeding duration [START_REF] Li | The validity and reliability of maternal recall of breastfeeding practice[END_REF]. Finally recruitment occurred during pregnancy and is not likely to be related to the maternal infant feeding choices during the four months after birth.

The breastfeeding rate at the maternity unit was higher in Nancy (82%), but not in Poitiers (60%), than the rates in the last National Perinatal Surveys in 2003 (63%) and 2010 (69%)(4). This reflects wide breastfeeding variations between centres. Actually, a breastfeeding promotion programme with a lactation consultant had been implemented in the early 2000"s in Nancy centre and could partly explain the higher breastfeeding rate at discharge in this centre.

The percentage of any breastfeeding decreased slowly in the first 3 weeks after discharge, suggesting that returning home after discharge did not strongly influence any breastfeeding.

Breastfeeding decrease was less marked in our cohort than those reported in previous French studies in the 1990"s [START_REF] Labarere | Initiation and duration of breastfeeding in obstetrical hospitals of Aix-Chambery (France)[END_REF][START_REF] Branger | Factors influencing the duration of breast feeding. A study of 150 women[END_REF], but close to those reported in recent studies UK [START_REF] Bolling | The Information Centre Part of the Government Statistical Service[END_REF] or US [START_REF] Blondel | Enquête Nationale Perinatale 2010: les naissances en 2010 et leur évolution depuis 2003[END_REF][START_REF] Li | Breastfeeding rates in the United States by characteristics of the child, mother, or family: the 2002 National Immunization Survey[END_REF]. Breastfeeding support after discharge from the maternity unit, such as home visits or peer support are not systematically proposed to all women in France and might be rare in our cohort. This lack of support during the first weeks after birth might contribute to the higher cessation of almost exclusive breastfeeding.

Socio-demographic factors related to breastfeeding initiation were also related to breastfeeding at 4 months of infant"s age. The effect of these factors were quite similar for any or almost exclusive breastfeeding. Infants with a foreign family origin were more likely to be mainly breastfed at 4 months than infants of French origin. The same trend has been shown previously for foreign mothers in France [START_REF] Bonet | Differential trends in breastfeeding according to maternal and hospital characteristics: results from the French National Perinatal Surveys[END_REF] and Sweden [START_REF] Wallby | Region of birth, income and breastfeeding in a Swedish county[END_REF], and among ethnic minorities in the UK [START_REF] Bolling | The Information Centre Part of the Government Statistical Service[END_REF]. These results support the hypothesis that the cultural background of the mother is an important predictor in both initiation and duration of breastfeeding.

We used maternal education and family income as indicators of the social situation of women. Our results are consistent with the literature that showed that women with a higher educational level breastfed longer [START_REF] Skafida | Juggling Work and Motherhood: The Impact of Employment and Maternity Leave on Breastfeeding Duration: A Survival Analysis on Growing Up in Scotland Data[END_REF][START_REF] Li | Breastfeeding rates in the United States by characteristics of the child, mother, or family: the 2002 National Immunization Survey[END_REF][START_REF] Lande | Breastfeeding at 12 months of age and dietary habits among breast-fed and non-breast-fed infants[END_REF][START_REF] Forste | Are US mothers meeting the Healthy People 2010 Breastfeeding Targets for initiation, duration, and exclusivity? The 2003 and 2004 National Immunization Surveys[END_REF]. In contrast, associations between breastfeeding duration and income are not consistent. If most of the studies had found higher breastfeeding duration among high income women [START_REF] Skafida | Juggling Work and Motherhood: The Impact of Employment and Maternity Leave on Breastfeeding Duration: A Survival Analysis on Growing Up in Scotland Data[END_REF][START_REF] Li | Breastfeeding rates in the United States by characteristics of the child, mother, or family: the 2002 National Immunization Survey[END_REF], some studies found higher odds among poor women breastfeeding for long periods (12 months) [START_REF] Wallby | Region of birth, income and breastfeeding in a Swedish county[END_REF] or a non significant association [START_REF] Forste | Are US mothers meeting the Healthy People 2010 Breastfeeding Targets for initiation, duration, and exclusivity? The 2003 and 2004 National Immunization Surveys[END_REF]. Probably, women with low income prefer breastfeeding because of the cost of formula. A British survey showed that few women choose breastfeeding for this reason, but low-income women in the UK receive tokens to buy baby milk or infant formula [START_REF] Bolling | The Information Centre Part of the Government Statistical Service[END_REF].Finally, our findings might result from the selection of a very particular sample of low-income women who had accepted to participate in the study and the follow-up.

Maternal employment at birth was not significantly associated with breastfeeding initiation in our cohort, after adjustment for confounding factors (data not shown). This result is in accordance to previous findings in France [START_REF] Bonet | Differential trends in breastfeeding according to maternal and hospital characteristics: results from the French National Perinatal Surveys[END_REF]. However, breastfeeding was longer among mothers who did not work before birth or return to work later. These findings are consistent with some studies [START_REF] Hawkins | The impact of maternal employment on breast-feeding duration in the UK Millennium Cohort Study[END_REF][START_REF] Guendelman | Juggling work and breastfeeding: effects of maternity leave and occupational characteristics[END_REF], but not all [START_REF] Skafida | Juggling Work and Motherhood: The Impact of Employment and Maternity Leave on Breastfeeding Duration: A Survival Analysis on Growing Up in Scotland Data[END_REF]. The present study provides additional evidence, showing that early return to work had a greater negative effect on almost exclusive breastfeeding than on any breastfeeding. These results could be partly explained by mothers who start introducing formula or stop breastfeeding in anticipation of their return to work.

Maternal leave provisions may impact the time of return to work, but their influence in breastfeeding duration is not easy to assess [START_REF] Galtry | The impact on breastfeeding of labour market policy and practice in Ireland, Sweden, and the USA[END_REF]. For example, in the mid-2000"s, British women benefited from a longer maternity leave compared to Dutch women [START_REF]OECD Family database[END_REF]. However, breastfeeding rates at 3-4 months of infant"s age were higher in the Netherlands than those in the UK(1), suggesting an important influence of economic, social and cultural factors [START_REF] Galtry | The impact on breastfeeding of labour market policy and practice in Ireland, Sweden, and the USA[END_REF].Furthermore the impact of recent policies towards extension of maternity leaves varied according to country. In UK this policy had little influence in the proportion of breastfeedingmothers who stopped breastfeeding before 6 months(9),whereas in Canada,extension of maternity leave, from a maximum of approximately 6 months to about 1 year, might have contributed to significant increases in the duration of any and exclusive breastfeeding [START_REF] Baker | Maternal employment, breastfeeding, and health: evidence from maternity leave mandates[END_REF].

We found no significant association between breastfeeding at 4 months and full-or part-time employment. This finding is in contrast with previous studies in Europe, which showed that women who worked part-time breastfed longer than women who worked full-time [START_REF] Skafida | Juggling Work and Motherhood: The Impact of Employment and Maternity Leave on Breastfeeding Duration: A Survival Analysis on Growing Up in Scotland Data[END_REF][START_REF] Hawkins | The impact of maternal employment on breast-feeding duration in the UK Millennium Cohort Study[END_REF], particularly for exclusive breastfeeding [START_REF] Lanting | Infant milk feeding practices in the Netherlands and associated factors[END_REF]. In addition, once mothers in our cohort had returned to work, few combined breastfeeding and work when compared with data from the UK [START_REF] Bolling | The Information Centre Part of the Government Statistical Service[END_REF]or the US [START_REF] Guendelman | Juggling work and breastfeeding: effects of maternity leave and occupational characteristics[END_REF]. It seems that, in general, women in France do not feel comfortable combining employment and breastfeeding or are unable to do so. In this context, the time mothers spend at work does not influence their ability to maintain breastfeeding.

There is a need to support mothers who wish to combine breastfeeding and work. In most European countries, legislation relating to breastfeeding for working mothers does not meet the minimum standards recommended by the International Labour Organization (ILO) 183 Convention [START_REF] Cattaneo | Protection, promotion and support of breast-feeding in Europe: progress from 2002 to 2007[END_REF]. For example, in France and the UK, there is no specific legislation for job protection and non-discrimination for breastfeeding mothers or standards regarding paid breastfeeding breaks. We do not know to what extent mothers in our cohort and their employers were aware of breastfeeding mothers" rights in the workplace in France(26) and whether mothers took lactation breaks, or had access to facilities to breastfeed or to express milk at work. Different strategies to combine work and breastfeeding have been described in the literature [START_REF] Fein | Success of strategies for combining employment and breastfeeding[END_REF],such as pumping at the workplace, child care available on site, allowing the mother to leave work to go to breastfeed the infant, and having the infant brought to the work site. However, no randomized trial was carried out to assess the effectiveness of workplace interventions, such as lactation breaks, and physical and childcare facilities, to promote breastfeeding [START_REF] Abdulwadud | Interventions in the workplace to support breastfeeding for women in employment[END_REF]. Nevertheless, observational studies suggest that workplace interventions (time off for baby"s illness, flexible time, part-time work)(9) contribute to breastfeeding among working mothers. In addition, lactation support at work have been recognised as costeffective by employers, decreasing for example absenteeism and staff turnover [START_REF] Shealy | The CDC Guide to Breastfeeding Interventions[END_REF].

In a society where breastfeeding is not the norm, it is difficult to support working mothers who wish to continue breastfeeding. In France, recent proposals in relation to breastfeeding have sought to enable more women to combine work and breastfeeding and to promote breastfeeding in childcare settings [START_REF] Boyer | Rapport parlementaire sur la prévention de l"obésité In: Mission sur la prévention de l"obésité[END_REF][START_REF] Salle | alimentation du nouveau né et du nourrisson[END_REF]. Recent attempts to increase maternity leave entitlements have failed [START_REF]Proposition de loi relative à la modernisation du congé maternité en faveur de la protection de la santé des femmes et de l"égalité salariale et sur lesconditions d"exercice de la parentalité[END_REF]. Increasing the length of maternity leave in France could allow more mothers who initiate breastfeeding to continue and could promote mother-infant bonding for all mothers, whether they are breastfeeding or not. However, it might not be easy to implement a longer maternity leave without adverse effects on mothers" professional careers or without increasing social disparities between women who need to return to work for financial reasons and those who can choose to extend their maternity leave.

Conclusion

Breastfeeding at 4 months of infant"s age was low in this study and failed to meet international(33) and French( 14) breastfeeding duration recommendations.

Sociodemographic characteristics of the mothers had a similar effect on any or almost exclusive breastfeeding duration. Return to work was a major predictor for stopping breastfeeding before 4 months, particularly for almost exclusive breastfeeding.In a society where breastfeeding is not the norm, it might be difficult to support working mothers who wish to continue breastfeeding. Specific actions need to be developed among mothers who return to work in the first months after their child"s birth and among employers and co-workers.

  weeks of gestation from the maternity wards of Poitiers and Nancy University hospitals between 2003 and 2006. Recruitment periods lasted 27 months in each centre. The EDEN cohort included 2002 women (969 in Poitiers, 1033 in Nancy) and represented an estimated 55% of the eligible women giving birth in the two centres. The EDEN study received approval from the ethics committee (CCPPRB) of Kremlin Bicêtre on 12 December 2002. Files have been declared to the "National Committee for Processed Data and

Funding:

  Sources of funding for the EDEN study: Fondation pour la Recherche Médicale (FRM), French Ministry of Research: IFR programme, INSERM Human Nutrition National Research Programme, and Diabetes National Research Programme (through collaboration with the French Association for Diabetes Research (ARD)), French Ministry of Health, French Agency for Environment Security (AFFSET), French National Institute for Population Health Surveillance (INVS), Paris-Sud University, French National Institute for Health Education (INPES), Nestlé, Mutuelle Générale de l"Education Nationale (MGEN), French speaking association for the study of diabetes and metabolism (ALFEDIAM), National Agency for Research (ANR non-thematic programme), National Institute for Research in Public health (IRESP: TGIR cohorte santé 2008 programme). Statistical analyses were carried out in complete independence of funders.

Figure 1 -

 1 Figure 1-Any and almost exclusive breastfeeding between discharge from maternity unit a d o ths of i fa t's age. Includes 1339 any breastfeeding and 1230 almost exclusive breastfeeding mothers who were breastfeeding at discharge from the maternity unit. I fa t's age in completed weeks or months.

Figure 2 -

 2 Figure 2-Any breastfeeding between discharge from maternity unit and 12 months of i fa t's age a o g wo e who worked duri g preg a y. Includes 979 mothers who worked during pregnancy and who were breastfeeding at discharge from the maternity u it. I fa t's age i co pleted weeks or o ths.

Acknowledgements

We thanks the participating families, our midwife research assistants (L Douhaud, S Bedel, B Lortholary, S Gabriel, M Rogeon, M Malinbaum) for data collection and P Lavoine, J Sahuquillo and G Debotte for checking, coding and data entry. We thank Y Akoli-Kodou,B de Lauzon-Guillain, and N Regnault for their collaboration with the construction of breastfeeding variables.

Ta le . Fa tors asso iated with reastfeedi g at 4 o ths of i fa t's age a o g others breastfeeding at discharge from maternity unit (n=1339)


Dorothée Kopp

Figuerola

Arthur Corn

Frédéric Santoul

Régis Céréghino
email: cereghin@cict.fr

Ecolab

Environmental effects related to the local absence of exotic fish

Keywords:

Given the extent of biological invasions in industrialized countries, our understanding of the determinants of overall patterns ofbiological invasions could gain most from consideration ofwhy exotic species are absent from sorne areas, rather than from distribution patterns of exotic species. Fish communities were sampled at 381 sites representing 221 rivers in the Adour-Garonne stream system (116 000 km 2 , SW France). Very few rivers were not colonized by exotic fish species, however, on a local basis, only 33% of the sampling sites hosted exotics. Using General Linear Modelling, we found that patterns of exotic fish (occurrence, number of species, proportion within assemblage) responded ta bath land-use and physical variables, whereas patterns of native fish only responded ta the local meso-scale characteristics of each stream reach from headwaters ta mouth. Ali fish communities were susceptible ta invasion regardless of native species richness, and higher native species richness did not decrease the opportunity for establishment by exotic species. The likelihood that exotic fish are absent primarily increased with elevation and with lower human influence upon the land caver, while human-impacted landscapes (agricultural and urban areas) were more likely ta host exotic fish and higher numbers of exotic species. In light of urban and agricultural development, our ability ta detect responses of exotic species ta landscape alterations using a combination of simple physical and land caver variables exemplifies a cast-effective technique for assessing areas at greater invasion risk in large stream systems.

Introduction

Introductions and invasions of alien species (including plants and animais) are a long-recognized problem [START_REF] Vitousek | Biological invasions as global environmental change[END_REF][START_REF] Lake | Invasion success of exotic plants in natural ecosystems: the role of disturbance, plant attributes and freedom from herbivores[END_REF], and our ability to demonstrate statistical patterns of invasion by exotic species has often been seen as a key to the success of future conservation and management projects [START_REF] Leung | Predicting invasions: propagule pressure and the gravity of allee effects[END_REF]. Specifically, much effort has been directed towards predicting habitat susceptibility to invasion, and forecasting locations at greatest risk of invasions [START_REF] Céréghino | Using self-organizing maps to investigate spatial patterns of non-native species[END_REF][START_REF] Holway | Edge effects of an invasive species across a natural ecological boundary[END_REF]. However, given the extent ofbiological invasions in industrialized countries, our understanding of the determinants of overall patterns of biological invasions may now gain most from consideration of why exotic species are absent from sorne areas, rather than from consideration of the distributions of exotic species.

Fresh waters are the recipient of many exotic species [START_REF] Moyle | Biological invasions of fresh water: empirical rules and assembly theory[END_REF] and the most frequently introduced freshwater organisms worldwide are fishes [START_REF] Gozlan | Introduction of non-native freshwater fish: is it ali bad?[END_REF][START_REF] Leprieur | Fish invasions in the world's river systems: when natural processes are blurred by human activities[END_REF]. Streams in particular have little immunity to exotic fishes [START_REF] Morton | The aquatic nuisance species problem: a global perspective and review[END_REF], typically introduced to enhance recreational fishing and aquaculture, or for culinary and aquarist purposes or even to reduce pest insects [START_REF] Keith | Atlas des poissons d'eau douce de France[END_REF]. Certainly due to fishing and/or economie concerns, their negative ecological effects (from the individual to the ecosystem leve!) are weil documented (e.g. [START_REF] Townsend | Individual, population, community, and ecosystem consequences of a fish invader in New Zealand streams[END_REF][START_REF] Koehn | Carp ( Cyprinus carpio) as a powerful invader in Australian waterways[END_REF], although sorne recent studies argued for negligible effects or even benefits for biodiversity (i.e. [START_REF] Gozlan | Introduction of non-native freshwater fish: is it ali bad?[END_REF]. Fish introductions in rivers are described since the Roman era [START_REF] Bajon | Origin and domestication of the wild carp, Cyprinus carpio: from Roman gourmets to the swimming flowers[END_REF], and intensified during the 19th century (Ailardi, 1984;[START_REF] Keith | Bilan des introductions de poissons d'eau douce en France[END_REF]. Surprisingly, sorne areas are still free of exotic fish (i.e. [START_REF] Sedell | Raie of refugia in recovery from disturbances: modern fragmented and disconnected river systems[END_REF] and seem to harbor relatively "pristine" conditions, in terms of species assemblage. This situation raises new questions about the factors that prevented, and still prevent, biological invasions in such areas.

In this study, we focussed on the Adour-Garonne stream system (SW France), which drainage basin is subjected to urbanization and extensive agriculture. Assuming that increasing anthropogenic impact increases the chances of successful dispersal and invasion by exotic fishes [START_REF] Lodge | Biological invasions: Lessons for ecology[END_REF], we hypothesized that exotic and native species would differ in their responses to physical ( e.g. elevation, stream order, distance from source, reach slope, air temperature) and land-use (e.g. urbanization, agriculture) variables. We predicted that exotic species richness broadly decreases with eleva-tian as anthropogenic pressures decline and streams become smaller. Specifically, we sought to produce explicit models which allow us to understand the relationships between the location of sites within the stream system, and the degree to which fish comrnunities have been invaded by exotic species.

2.Method5

Study area and data collection

The Adour-Garonne stream system (south-western France) has a 116 000 km 2 drainage basin. During the last decades, the basin and its rivers have been intensively studied for various ecological aspects; a thorough description of the system was given in [START_REF] Tockner | Rivers of Europe[END_REF]. We selected 381 sampling sites (Fig. 1) ranging from2 to 1800 rn a.s.l., thus representing 221 streams from high mountain, plain, and coastal areas.

Ali sites were sampled once by electrofishing during law-flow periods between 2002 and 2007. Two-pass removal sampling was used whenever possible, by wading in smaller rivers and by boat in the larger ones. Forty eight fish species were found to occur in the Adour-Garonne basin (Table 1 ), among which 17 were exotic species (according to Keith andAllardi, 1997, 2001;[START_REF] Persat | La répartition géographique des poissons d'eau douce en France : qui est autochtone et qui ne l'est pas 7[END_REF]. The biological variables assigned to each site for this study were: the occurrence (presence or absence) of exotic species, percentage of exotic species, number of exotic species, and the number of native fish species.

0 0 o . 0 • •• 0 • g a .Il • 0 • 0 • •• • • • • • , • • • • •• 500 • • • 200 If •• ••
For each site, a Geographie Information System {GIS, Mapinfo professional 7.8) was used to delineate a geographical buffer zone representing a 1 000 rn-radius centered on the site. This size is weil suited to assigna land-caver influence to each site (see also Cornpin and Céréghino, 2007) and falls within that of the "Reach Buffer" sensu [START_REF] Allan | Landscapes and riverscapes: the influence of land-use on stream ecosystems[END_REF], i.e., a buffer of 100 rn to severa! hundred meters in width on each bank and sorne hundreds of meters to a kilometer in length. Sampling sites were then characterized using six physical variables, and three land-caver variables intended to account for anthropogenic pressure. The physical variables were elevation above sea leve! (a.s.l., rn), slope ('L), stream arder, distance from the source (km), drainage basin area (km 2 ), and maximum air temperature in june (°C, i.e. the mean of maximum temperatures recorded by the French Meteorological Services in early summer during the whole sampling period). The three land-caver variables were percent area within a buffer zone covered by forest (areas occupied by forest and woodlands with native or exotic coniferous and/or deciduous trees; scrub and herbaceous vegetation associations), urban development (industrial, commercial and transport units; artificial and non-agricultural vegetated areas), and agricultural area (arable lands, permanent crops and pasture). Digital land caver information was obtained from the CORINE land-cover database for Europe (CLC, 2000, European Environment Agency http:lf www.eea.europa.euf; see also [START_REF] Cruickshank | Application of CORINE land cover methodology to the UK. Sorne issues raised from Northern Ireland[END_REF]. This database was generated from orthorectified satellite images and provides thematic GIS map layers including up to 44land-cover classes with a mapping scale of 1: 1 00 000. These nine variables Table 1 Ust of the 48 fish species considered in this study with their status (N = native, E = exotic E) and their percentage of occurrence (number of sites where the species was foundjtotal number of sites were chosen because they characterize the location of sampling sites within the stream system and within the regional landscape mosaic, and they are easy to describe using a GIS. The use of simple variables in a successful final mode! could thus reduce the effort and cost of data collection for water management applications.

• • • 0 • • • i !!1 0 • 0 0 0 • • • • • • 0 0 • • • 1 • • ,. • 0 •• • 0 • 0 .::1 • • ji o • o • • • • • • • 0 • 0 • • • . - • • 0 0 • •• • N 1 lOO km

Data analyses

To analyze the relationships between environmental variables and occurrence of exotic species, percentage of exotic species, number of exotic species, and the number of native fish species, we used Generalized Linear Modelling, including river basin as a random factor to control for the possible non-independence of sampling points coming from the same basin. When the dependent variable corresponded to count data (number of exotics or number of native species) we used Poisson distributed errors and the log link to fit the models. When the dependent variables corresponded to occurrence data or proportions (presence of exotics or number of exotics out of total number of fish species) we used binomial dis-tributed errors and the logit link to fit the models. Ali analyses were done with the GLIMMIX procedure in SAS 9.2. (SAS Institute 2009). Models were fitted using Laplace approximation which is more accurate than Penalized quasilikelihood [START_REF] Bolker | Generalized Iinear mixed models: a practical guide for ecology and evolution[END_REF], and based on likelihood. Significance of random effect was tested by likelihood ratio test using the covtest statement. Environmental variables were log transformed to fit a normal distribution (elevation, distance, slope and drainage area) or ranked when not normalized by usual transformations (temperature, % urban and % agricultural area); see [START_REF] Conover | Rank transformations as a bridge between parametric and nonparametric statistics[END_REF]. Stream order and number of native species fitted a normal distribution. As land-use was classified in three exclusive categories (%urban, agricultural and forest areas), a strong colinearity existed between the three variables. For this reason, only two of the categories were included in further analyses (%urban and% agricultural areas), but statistically significant relationships of the same sign for both variables must also be interpreted as a negative relationship for the third one.

Results

Fish were present in ali sampling sites, with exotic species occurring at 129 sites out of 381. The examination of simple occurrence data of exotic fishes on a geographical map (Fig. 1) suggests that most headwater sites are depleted in exotic species compared to those sites located in plain areas. This is particularly true of the Pyrenees Mountains (south of the Adour-Garonne system) and the Massif Central Mountains (east of the system). However, the drainage basin, which is located in the northwestern plain area, is also devoid of exotic species. The variance explained by the exotic species occurrence mode! was 48.6%. The occurrence of exotic species was positively correlated with the number of native species (p = 0.0004; Table 2) and negatively correlated with elevation and slope (p < 0.0001 and p = 0.007, respectively). In other words, the likelihood that exotic species are locally absent increased with decreasing numbers of native species and increasing elevation. The proportion of exotic species within local assemblages was negatively correlated with slope and elevation (p = 0.01 and p = 0.002, respectively) and it was positively correlated with temperature, stream order, urbanization and agriculture (p < 0.0001, p = 0.04, p = 0.02 and p = 0.02, respectively ). The proportion of exotic species decreased with elevation, but increased with temperature and anthropogenic factors.

The exotic fish richness mode! explained 38.5% of the total variance in numbers of exotic species. The number of exotic species was influenced both by natural factors and anthropogenic ones. Slope and elevation were negatively correlated with the number of exotic species (p = 0.002 and p = 0.01, respectively; Table 2). Temperature was positively correlated with exotic species richness (p < 0.0001 ), as weil as urbanization (p = 0.02). By contrast, landuse variables did not correlate significantly with the number of native species, meaning that topographical zonation overcame the anthropogenic influence. The native fish richness mode! explained 23.6% of the total variance in numbers of native species. Slope and elevation were negatively correlated with the number of native species (p < 0.0001 and p = 0.0003, respectively), whereas temperature and distance from the source were positively correlated with native species richness (p = 0.02 and p < 0.001, respectively; Table 2).

Discussion

Throughout the world, environmental policies aiming at preserving the biological diversity of terrestrial and/or aquatic ecosystems heavily rely on action plans for the delineation of zones of ecological interest (which concentrate rare and/or threatened species, or which have patrimonial values -see e.g., the Natura 2000 network of the European Union). Biological invasions often form part of such frameworks through more specifie projects intended to enhance our theoretical knowledge of interactions between native and exotic species (e.g. Cucherousset et al., 2008a), and/orto propose practical decision tools for managers faced with sets of exotic species that generate local and/or regional nuisance. These policies therefore have an impact on a wide range of people and activities such as water consumers and recreational users, agriculture, industry and business activities. Environmental managers generally ask for explicit schemes such as distribution patterns that allow them to identify areas at greater ecological risk. If threats associated with economy-oriented projects align with zones deprived of exotic species, this could add new impetus to calls for freshwater conservation policies. Among the questions that are posed to the scientific experts, the most common ones are: (1) which areas within a regional system contain exotic species? and (2) which environmental variables explain the presence of exotic species? In the Adour-Garonne stream system (SW France), very few ri vers were not colonized by exotic fish species. However, on a local basis, only 33% of the sampling sites hosted exotic fish species. Paradoxically, the local absence of exotic species in such invaded systems is thus more intriguing than the occurrences.

With a large number of explanatory variables, one may overfit the models so that they perform weil in the context of the dataset used to generate them, but fail to be robust when used elsewhere [START_REF] Rushton | New paradigms for modeling species distributions[END_REF]. The variance explained by our GLMs was about 48% for the exotic species occurrence model, and 38.5% for exotic species richness, although we used a limited number of variables. The variance explained by the native fish and proportion of exotic fish models was lower, but still about 24-25%. The suggested schemes are thus relevant, and should perform weil in other areas. Our results showed that patterns of exotic fish (occurrence, number of species, proportion within assemblage) responded to both land-use and physical variables, whereas native fish responded to the gradient of physical characteristics of streams from headwaters to mou th. The species richness of native fish gradually increased from headwaters to plains as a result of downstream addition of species, suggesting that anthropogenic alterations of the riparian and watershed landscapes did not override geomor-phological controls on the distribution of native fish (see Cucherousset et al. (2008b) for a detailed analysis of patterns for native fish in our study system). The likelihood that exotic fish are absent increased with elevation and reach slope, while the proportion of exotic species within assemblages decreased with decreasing urbanization and agriculture. The positive correlation between occurrence of exotic species and native species richness indicated that ali communities were susceptible to invasion regardless of native species richness (see also [START_REF] Moyle | Biological invasions of fresh water: empirical rules and assembly theory[END_REF][START_REF] Gido | Invasion of North American drainages by alien fish species[END_REF], and that higher native species richness did not decrease the opportunity for establishment by exotic species [START_REF] Mack | Biotic invasions: causes, epidemiology, global consequences, and control[END_REF]. The relationship between native species richness and invasion success is intensely debated. Sorne models suggest that species richness should limit the capacity of new species to establish in an area due to the saturation of the available niches [START_REF] Elton | The Ecology of Invasions by Animais and Plants[END_REF][START_REF] Case | Invasion resistance arises in strongly interacting species-rich mode! competition communities[END_REF][START_REF] Tilman | The ecological consequences of changes in biodiversity: a search for general principles[END_REF][START_REF] Kennedy | Biodiversity as a barrier to ecological invasion[END_REF]. However, many observational studies suggest the opposite, the richness of native and exotic species being correlated [START_REF] Gilbert | Invasibility and abiotic gradients: the positive correlation between native and exotic plant diversity[END_REF][START_REF] Perelman | Habitat stress, species pool size and biotic resistance influence exotic plant richness in the Flooding Pampa grasslands[END_REF] or, as in this study, exotics being present in the areas with greater native species richness.

Mountain streams may represent harsh environments which are physically stressful (higher river competence and erosive forces generated through the combination of slope with other variables such as water depth and current velocity, snowmelt floods, etc.), so that a smaller proportion of potential colonizers could become established [START_REF] Gido | Invasion of North American drainages by alien fish species[END_REF]. Sorne sites located in the plain, especially in the northwestern area of the watershed, presented very low proportions of exotic fish. However, we assume that these particular plain sites present lower opportunities for fish introduction because of the lower human influence in this sub-system [START_REF] Tockner | Rivers of Europe[END_REF]) (see below), rather than because of fast flowing and fluctuating environments.

Mountain sites are Jess subject to human influence, and have usually not been subjected to high levels of introductions. We found that the proportion of exotic species in the fish assemblage and the local numbers of exotic species were positively correlated with the extent of urbanization. According to our GLM results, this pattern did not result from drops in native species richness with increasing anthropogenic influence, but actually from increases in numbers of exotic species with urban development. Human-impacted landscapes ( characterized here by higher proportions of urban and/or agricultural areas) are more likely to host exotic fish and higher numbers of exotic species [START_REF] Leprieur | Fish invasions in the world's river systems: when natural processes are blurred by human activities[END_REF], and this was true for the Adour-Garonne stream system. According to [START_REF] Mckinney | Effects of human population, area, and time on non-native plant and fish diversity in the United States[END_REF], human population size is a good predictor of exotic fish species richness. Similar observations were reported for plants [START_REF] Whitney | A quantitative analysis of the flora and plant communities of a representative Midwestern U.S. town[END_REF], mammals [START_REF] Hansen | Effects of exurban development on biodiversity: patterns, mechanisms, and research needs[END_REF], and birds [START_REF] Germaine | Relationships among breeding birds, habitat, and residential development in Greater Tucson, Arizona[END_REF][START_REF] Donnelly | Importance of reserve size and landscape context to urban bird conservation[END_REF]). Therefore, both literature data and own our study support the conclusion that exotic species associated with humans predictably colonize ecosystems as agricultural and/or urban land cover increases.

In conclusion, whilst exotic species were predictably more likely to be absent from headwater streams subjected to low anthropogenic influence, we demonstrated that the number and proportion of exotic species responded to broad land-cover categories such as "agricultural area" and "urban area", and that the 1000 rn-radius buffer zone adapted from [START_REF] Allan | Landscapes and riverscapes: the influence of land-use on stream ecosystems[END_REF] was appropriate to detect changes in number and proportion of exotic species in a large stream system. Since the challenge of recent applied research is to assess models having the broadest capability of predicting spatial patterns of community organization (including native and exotic species ), we suggest that the combination of simple physical and land-cover variables obtained from thematie GIS map layers should be relevant to identify and to delineate areas of concern in integrated management of biological invasions at watershed levels. Moreover, our study implicitly supports the idea that action plans should be designed at a landscape scale [START_REF] Ward | Riverine landscapes: biodiversity patterns, disturbance regimes, and aquatic conservation[END_REF]. In light of urban and agricultural development, our ability to detect responses of exotic species to landscape alterations exemplifies a cast-effective technique for assessing areas at greater invasion risk in large stream systems.

Fig. 1 .

 1 Fig. 1. The Adour-Garonne basin. and location of the 381 sampling sites. Filled squares denote locations where at !east one I!XIltic fish species was present. white squares denote locations where I!XIltic fish are absent.

  ).

	Species	Status	Occurrence {%)
	Salmo trutta faria	N	0.63
	Phoxinus phoxinus	N	0.60
	Gobiogobio	N	0.59
	Barbatula barbatula	N	0.49
	Leuciscus cephalus	N	0.46
	Rutilus rutilus	N	0.33
	Anguilla anguilla	N	0.29
	Barbus barbus	N	0.28
	Albumus albumus	N	0.26
	Lampetra planeri	N	0.20
	Lepomis gibbosus	E	0.20
	Perca fluviatilis	N	0.19
	Leuciscus leuciscus	N	0.18
	Salmo salar	N	0.17
	Cottus gobio	N	0.14
	Esox ludus	N	0.08
	Chondrostoma taxostoma	N	0.08
	Cyprinus carpio	E	0.08
	Abramis brama	N	0.07
	Carassius sp.	E	0.07
	Silurus glanis	E	0.07
	Tinca tinca	N	0.07
	Ameiurus melas	E	0.06
	Scardinius erythrophtalmus	N	0.06
	Rhodeus amarus	N	0.06
	Micropterus salmoides	E	0.06
	Blicca bjoerkna	N	0.06
	Gymnocephalus cemua	E	0.06
	Onchorhynchus mikiss	E	0.04
	Pseudorasbora parva	E	0.03
	Sander ludoperca	E	0.03
	Barbus meridionalis	N	0.03
	Petromyzon marinus	N	0.03
	Gasterosteus aculeatus	N	0.02
	Pungitius pungitius	N	0.02
	Gambusia affinis	E	0.02
	Pachychilon pictus	E	0.02
	Thymallus thymallus	E	0.01
	Platichthys jlesus	N	0.01
	Hypophtalmichthys molitrix	E	0.01
	Salve linus fontinalis	E	0.01
	Chelan labrosus	N	0.01
	Leuciscus burdigalensis	N	0.01
	Albumo bipunctatus	E	<0.01
	Leucaspius delineatus	E	<0.01
	Alosa alosa	N	<0.01
	Cobitis taenia	N	<0.01
	Leuciscus soufia	N	<0.01

Table 2

 2 Models analyzing the patterns of fish distributions using four biological indicators; occurrence and proportion of exotic fish species, number of native and exotic species. Only variables with p < 0.05 are interpreted as statistically significant. Deviance of the null models, dispersion values ( <1> =deviance of the final modeljdegrees of freedom) and percentage of variance explained are given.

		Effect
	Occurrence of exotic species	Elevation
		Distance source
		Slope
		Drainage area
		Temperature
		Urbanization
		Agriculture
		Stream order
		Number of native species
		Deviance
		<1>
		Variance explained
	Proportion of exotic species	Elevation
		Distance source
		Slope
		Drainage area
		Temperature
		Urbanization
		Agriculture
		Stream order
		Deviance
		<1>
		Variance explained
	Number of native species	Elevation
		Distance source
		Slope
		Drainage area
		Temperature
		Urbanization
		Agriculture
		Stream order
		Deviance
		<1>
		Variance explained
	Number of exotic species	Elevation
		Distance source
		Slope
		Drainage area
		Temperature
		Urbanization
		Agriculture
		Stream order
		Deviance

<1>

Variance explained

Acknowledgements

We wish to thank the Office National de l'Eau et des Milieux Aquatiques, and more specifically Dr. N. Poulet, for providing us with the fish data.