domain: sde qfin sdv shs

text:
Moriah B Bostian

Pierre Dupraz
email: pierre.dupraz@rennes.inra.fr

Jean Joseph Minviel

Working Paper SMART -LERECO N°15-12 Production effects of wetland conservation: evidence from France

Keywords: wetlands, agri-environmental schemes, agricultural production, conservation payments, France JEL classification: Q15, Q51, Q53, Q58 zones humides, mesures agro-environnementales, production agricole, élevage, paiements pour services environnementaux, France Classification JEL : Q15, Q51, Q53, Q58

This study takes a production function approach to examine the effects of farm wetland area for a set of producers in the Limousin region of France. By combining data from a recent survey of regional wetland areas with detailed farm-level production panel data, we find that maintaining wetland areas poses significant costs to farmers, in terms of foregone production value. These results help to explain the relatively low participation rate in agri-environmental schemes targeted to wetlands by farmers in this region. This represents a new application of the production function approach to estimate the cost of maintaining wetlands on working agricultural land, and is one of few studies to examine agricultural wetland costs outside of the US. This framework could be used to further inform payment incentives for agrienvironmental schemes more generally.

The SMART-LERECO Working Papers are meant to promote discussion by disseminating the research of the SMART and LERECO members in a preliminary form and before their final publication. They may be papers which have been accepted or already presented in a national or international scientific conference, articles which have been submitted to a peer-reviewed academic journal, or chapters of an academic book. While not peer-reviewed, each of them has been read over by one of the scientists of SMART or LERECO and by one of the two editors of the series. However, the views expressed in the SMART-LERECO Working Papers are solely those of their authors.

Production effects of wetland conservation: evidence from France 1 Introduction

Wetlands provide a number of important and widely-known services, including support for biodiversity, improved hydrological function, recreation and scenic amenities. Many of the most critical wetlands in Europe and the United States extend across rural landscapes, where agriculture in particular benefits from greater flood control, water purification and biological pest control [START_REF] Ma | Valuation of ecosystem services from rural landscapes using agricultural land prices[END_REF]. At the same time, agricultural runoff of nitrogen fertilizers and animal waste, as well as increased drainage and channelization of agricultural lands, pose some of the greatest threats to wetland condition [START_REF] Brinson | Temperate freshwater wetlands: types, status, and threats[END_REF]. Given these linkages, designing policies to protect rural wetlands requires some understanding of agricultural land use decisions. In the U.S., the primary rural wetland policies, the Wetlands Reserve Program (WRP) and the Conservation Reserve Program (CRP), mainly offer incentives to farmers to take wetland areas out of production in the form of easement payments. In contrast, the various agri-environmental schemes (AES) employed by EU member states rely more on incentives to engage in less-damaging production practices, without necessarily ceasing production activities [START_REF] Baylis | Agri-Environmental policies in the EU and United States: A comparison[END_REF]. We examine the cost to farmers of maintaining wetlands on land used in production, in this case for a set of cattle and dairy farmers in the Limousin region of France. The Limousin, in the center southwest, is considered France's heartland and is known as the "area of 1,000 lakes." The region is also recognized for its high quality livestock production, supported by grazing on grassland areas, and its wetlands provide critical habitat to numerous freshwater fish and waterfowl species. AES participation in schemes targeted to wetlands by farmers in this region is relatively low (less than 10 percent), suggesting that current AES payments may not adequately compensate farmers for the associated compliance costs of wetland maintenance. Adequate compensation requires knowledge of these costs, and from a policy perspective, a better understanding of participation cost is important for designing effective AES incentives. To estimate the costs of maintaining wetland areas to farmers, we take a production function approach to directly model the effects of farm wetland area on farm output. We apply this framework to panel data on farm-level production, similar in format to the EU Farm Accountancy Data Network (FADN) standards, coupled with data from a unique survey of agricultural wetlands conducted in the region. Our results indicate that average costs to farmers of maintaining wetland areas exceed prevailing wetland AES payments in this region, which is consistent with current low observed wetland AES participation rates. More generally, this study contributes to the agri-environmental policy literature in two important ways. First, we estimate the opportunity cost to farmers of maintaining wetlands on land in production, rather than on retired lands. Second, our use of detailed farm production data allows us to estimate this cost directly, in terms of foregone production value, rather than implicitly through agricultural land values. We provide some background on agricultural wetlands and the French AES in the next section. We discuss the production model and identification strategy in Sections 3 and 4, and then present our empirical results in Section 5.

Background

The response to conservation incentives for agricultural wetlands depends largely on the relative benefits and costs of these policies at the farm level. On-farm benefits of maintained wetlands include water regulation, soil drainage, natural pest control from increased biodiversity, as well as recreational and aesthetic services [START_REF] Ma | Valuation of ecosystem services from rural landscapes using agricultural land prices[END_REF]. The wetlands valuation literature is extensive1 , but includes relatively few studies that value wetland services to agriculture, of which the focus is primarily on wetland services to agriculture in the developing world [START_REF] Acharya | Valuing groundwater recharge through agricultural production in the Hadejia-Nguru wetlands in northern Nigeria[END_REF][START_REF] Nordblom | Upstream demand for water use by new tree plantations imposes externalities on downstream irrigated agriculture and wetlands[END_REF]. Wetland costs to agriculture arise primarily from diminished production capacity on wetland areas [START_REF] Heimlich | Costs of an Agricultural Wetland Reserve[END_REF]. In addition to reduced arable land area year round, natural draining and filling cycles can limit production activities [START_REF] Shultz | Implicit Prices of Wetland Easements in Areas of Production Agriculture[END_REF], and irregularly-shaped or widely-dispersed wetlands can impose nuisance costs related to navigating machinery around each hydrated area [START_REF] Gelso | Farmers' Perceived Costs of Wetlands: Effects of Wetland Size, Hydration, and Dispersion[END_REF][START_REF] Cortus | The Economics of Wetland Drainage and Retention in Saskatchewan[END_REF]. The U.S. Fish and Wildlife Service (FWS) sponsored two early studies to value wetland costs to agriculture [START_REF] Brown | A study of the impact of the Wetlands Easement Program on Agricultural Land Values[END_REF][START_REF] Brown | Measuring the impact of Wetland Easements on Land Values in North Dakota, South Dakota and Minnesota. Internal report prepared for the U.S. Fish and Wildlife[END_REF] by measuring the effect of wetland easements on agricultural land values. The FWS then used the results of these studies to set location-specific easement payments. Despite their continued use in practice, [START_REF] Shultz | Implicit Prices of Wetland Easements in Areas of Production Agriculture[END_REF] note several potential flaws in the Brown studies, including an apparent double-counting of wetland acreage, small sample size and omitted variables bias related to non-eased wetland acreage. The authors address these weaknesses in their own study by working with a much larger set of land sales data, including agricultural revenues attributed to sale-specific cropping patterns and soil quality, and using geographical information system to control for hydrologic condition. They find that an additional acre of non-eased permanent wetlands decreased average sale price by 40%, or $161 per acre at the time, while non-eased temporary wetlands had no statistically significant effect. Their estimated effect is greater for lands with easements in place, with a 79% or $321 per acre decrease for eased permanent wetlands, but again, no significant effect for eased temporary wetlands. This lack of a land value effect for temporary wetlands across easement states suggests a lesser, or perhaps even ambiguous, effect of temporary wetlands on production capacity. [START_REF] Lawley | Changes in Implicit Prices of Prairie Pothole Habitat[END_REF] uses farm sales data from the prairie pothole region in Manitoba for the 20 year period 1990-2009 to estimate the change in implicit wetland costs over time. For the midrange 1998-2001 period, the author finds that a 1% increase in wetland acreage lowers per acre sale price by 1.15%, which translates to a $456/acre discount for an average size parcel. Pooling the 1990-1993 period with the 2006-2009 period, the author estimates that implicit wetland costs increased by 40% over the 20 year period, and that this increase in wetland cost holds similarly for the 25%, 50% and 75% quantiles. Estimated implicit prices outweigh real offer payments under Manitoba's Wetland Restoration Incentive Program (WRIP) for the majority of sites, which Lawley notes, suggests the WRIP is primarily targeting low valued land in the region. [START_REF] Gelso | Farmers' Perceived Costs of Wetlands: Effects of Wetland Size, Hydration, and Dispersion[END_REF] allow for ambiguous wetland effects related to total wetland area and frequency of hydration, but an unambiguously negative effect for wetland dispersion, in an expected utility of income modeling framework. They measure the cost of wetlands as the certainty equivalent of producing on a field with zero wetland area. The authors carry out a contingent valuation survey for a large set of Kansas farms to estimate perceived costs for these wetland characteristics. They find that an additional percentage of wetland coverage increases perceived costs by $0.60 per acre, and that if a given wetland area is dispersed into four separate areas rather than one, the additional cost is $4.23 per acre. This dispersion cost increases to $10.47 per acre for 16 separate wetland areas. They also find that hydration frequency increases wetland cost, by roughly $1.00 per acre for wetlands that hydrate with more than 20% frequency. [START_REF] Yu | An economic analysis of landowners' willingness to adopt wetland and riparian conservation management[END_REF] directly survey farmers' willingness to accept (WTA) for enrollment in a hypothetical 10-year wetland and riparian conservation management program, using a closedended accept/reject bid payment question format. Employing a probit modeling framework, they estimate an average WTA of 75.32 CAD per ha. In addition to farm characteristics, such as size and soil quality, they find that farm operator attitudes and experience with wetlands significantly affect the conservation decision. For instance, farmers who believe that wetlands provide positive net benefits and those who already maintain riparian areas are more likely to enroll for a given payment level, while those who believe that wetlands have aesthetic value, increase operation costs, or are important for wildlife are less likely to enroll. Production losses associated with on-farm wetlands can also influence the wetland conversion decision. In their simulation of potential exemptions under the US Swampbuster program, [START_REF] Claassen | Estimating the effects of relaxing agricultural land use restrictions: Wetland delineation in the Swampbuster Program[END_REF] base the decision to convert wetland area on whether the net present value of expected returns to the resulting crop production outweighs the total costs (including opportunity cost) of conversion. Working with a national dataset for agricultural wetlands and commodity production, they estimate that under baseline prices, conversion would be profitable for 5.6-12 million acres. They find that most conversion of cropped wetland area would take place in the Prairie Pothole region, and that conversion estimates are especially sensitive to commodity price changes. [START_REF] Cortus | The Economics of Wetland Drainage and Retention in Saskatchewan[END_REF] incorporate dynamic stochastic commodity prices and yields, weather variables and drainage time constraints into their simulation of wetland conversion in the Saskatchewan province of Canada. They estimate average annual private net benefits of conversion ranging from $27.76/ha of wetland drained on a 4 quarter farm to $40.66/ha for a 20 quarter farm, suggesting increasing returns to farm size for wetland conversion. They also find that net benefits of conversion are greater for positive nuisance costs, but that the effects of constant versus increasing nuisance cost scenarios are virtually the same. The authors note significant variability in their results, which they attribute to the use of stochastic price and yield data. Turning to AESs in France, Chabé-Ferret and Subervie (2013) model the participation decision for several AESs related to surface water quality and employ a differences-in-differences with matching framework to estimate the effects of AES participation on farm practices. They find that AESs have increased the planting of cover crops by an average of 10 ha per farm, and the average use of grass buffer strips by 240 meters for participants. They also find that AESs increased conversion to organic farming by approximately 46 ha and account for 90% of all increases in organic farming from 2000-2005. Combining these with existing benefit estimates and reported program costs, they find that AESs for cover crops are likely not cost effective, due in part to large estimated windfall effects, and that buffer strips are likely only cost effective for larger watersheds. In contrast, they find that AESs for conversion to organic farming are likely highly cost effective. The AES measures targeted to wetlands in our study region include restrictions to tillage and drainage of wetland areas, fertilizer use, winter grazing, and animal density. Previous studies have estimated wetland production costs implicitly using agricultural land sales data, or added them conceptually to a choice modeling problem. We estimate them directly from farm-level production data using a production function framework, which also represents a new application of this approach to agricultural wetland costs in the EU. We outline the production function and estimation methods in the next sections, and then present results from our application to farmers in the Limousin region.

The Production Model

We model farm output as a function of productive inputs (e.g., land, labor, fertilizer, and capital), on-farm wetland area, and other farm characteristics such as age, commercial designation and subsidy participation. Let y it be production output for farm i in time period t, so that

y it = f (x it , w it , h it ), (1)
where x it is a vector of productive inputs, w i,t is on-farm wetland area and h it is a vector of farm characteristics. A Cobb-Douglas specification of the production technology yields the econometric model,

lny it = lnx it β 1 + w it β 2 + h it β 3 + it , (2)
where it is composed of unobserved farm-level fixed effects, a i , and a time-varying disturbance, u it .

Our output variable y it represents farm annual value of final product. Conventional agricultural inputs in x it include utilized land area measured in hectares and labor measured in hours, as well as total expenditures for fertilizer, seed, pesticide, feed, fuel, animals and capital asset value. We specify wetland area, w it , as percent of total utilized agricultural area. Farm characteristics in h it include the share of rented land area, family farm designation (as opposed to commercial) and farm operator age, as well as whether or not the farm lies in an area designated as mountainous or 'less favorable'. Less favored status is granted by the government to areas with inferior growing conditions, such as steep slopes or poor soil quality. We also include total annual coupled and decoupled subsidy payments under the EU common agricultural policy (CAP). Decoupled subsidy payments have been found to have small, but significant positive acreage effects in the US [START_REF] Goodwin | Are "Decoupled" Farm Program Payments Really Decoupled? An Empirical Evaluation[END_REF] and largely negative effects on total factor productivity for crop production in France [START_REF] Mary | Assessing the Impacts of Pillar 1 and 2 Subsidies on TFP in French Crop Farms[END_REF]. The coefficient on wetland area provides a measure of the effect of on-farm wetland area on farm value of final product, controlling for conventional productive inputs and other farm characteristics. Given previous estimates of implicit agricultural wetland cost [START_REF] Shultz | Implicit Prices of Wetland Easements in Areas of Production Agriculture[END_REF][START_REF] Lawley | Changes in Implicit Prices of Prairie Pothole Habitat[END_REF], we expect this coefficient to be negative. The production function approach to environmental valuation has been applied to various environmental-production relationships. [START_REF] Barbier | Valuing Environmental Functions-Tropical Wetlands[END_REF]2007) develops a production function methodology to value tropical wetlands as an input into fisheries production. [START_REF] Acharya | Valuing groundwater recharge through agricultural production in the Hadejia-Nguru wetlands in northern Nigeria[END_REF] use this framework to value wetland-supported groundwater recharge as an input into irrigated agricultural production. [START_REF] Klemick | Shifting cultivation, forest fallow, and externalities in ecosystem services: Evidence from the Eastern Amazon[END_REF] specifies a log-linear production function with spatial weighting to value the on-farm benefits and off-farm externalities of agricultural land fallowing. [START_REF] Laukkanen | Environmental and Production Cost Impacts of No-till in Finland: Estimates from Observed Behavior[END_REF] use a translog cost function to estimate the production cost of conservation tillage practices, and to then derive input factor demands for fertilizer and chemical use. [START_REF] Laukkanen | Evaluating greening farm policies: a structural model for assessing agri-environmental subsidies[END_REF] use a similar framework to model producer land, fertilizer and pesticide use decisions in response to AES payment rates.

Identification Strategy

In our study case, farm production varies from year to year, while farm characteristics such as commercial or less favored area status, and importantly, our variable of interest, on-farm wetland area do not change over the three-year study period. A fixed effects (FE) specification effectively differences these time-invariant effects away, and would only allow us to identify the time-varying effects of the conventional production inputs. We could include the time-invariant factors in a random effects (RE) specification as long as both observed time-invariant factors (wetland area, commercial and less favored area status) and time-varying factors (land in use, labor, etc.) are exogenous to the unobserved fixed effects [START_REF] Mundalk | On the pooling of time series and cross-section data[END_REF]. This highlights the well known tradeoff between FE and RE [START_REF] Hausman | Panel Data and Unobservable Individual Effects[END_REF]Baltagi et al., 2003). [START_REF] Hausman | Specification tests in econometrics[END_REF] tests of the difference between the FE and RE estimators indicate the RE strict exogeneity assumption is indeed violated in this case.

In order to exploit the panel nature of the production data without discarding information on the variable of interest, farm wetland area, we employ a Hausman-Taylor (HT) [START_REF] Hausman | Panel Data and Unobservable Individual Effects[END_REF] estimation framework. The HT estimator relaxes the often problematic assumption of zero correlation between the included explanatory variables and the fixed effects a i made in the standard RE model, while also accommodating observed time-invariant explanatory effects [START_REF] Greene | Econometric Analysis, Fifth Edition[END_REF]. In this framework, both time variant and invariant effects are separated into those that are correlated with the unobserved fixed effects a i , commonly referred to as the endogenous variables, and those that are uncorrelated with a i , referred to here as the exogenous variables. It is important to note that endogeneity in this framework refers to correlation between the observed explanatory variables and the unobserved fixed effects a i , but not the time-varying disturbance, u it .

The HT specification uses deviations in the time-varying variables within the model to instrument for the time-invariant variables. This allows for identification of the time-invariant variables without imposing the strict exogeneity assumption of RE. A HT specification of our log-linear production model in (2) yields

lny it = x it β + z i γ + a i + u it , (3)
where x it represents the time-varying production factors, including the conventional inputs in log form, and z i the time-invariant factors, including farm wetland area. We can decompose

x it = [x 1 it x 2 it ] and z i = [z 1 i z 2 i ],
where (x 1 it , z 1 i ) denote the (K 1 , L 1 ) exogenous variables (assumed uncorrelated with a i ) and (x 2 it , z 2 i ) denote the (K 2 , L 2 ) endogenous variables (assumed correlated with a i ).

The HT estimator makes use of the usual FE transformation of the time-varying variables to construct consistent estimators of β. Given its exogeneity to a i , z 1 i serves as an instrument for itself, while the mean of x 1 it , x1 serves as an instrument for z 2 i . This makes K 1 ≥ L 2 a necessary condition for identification. The instrumental variables for the HT model are thus

[(x 1 it -x1 ), ((x 2 it -x2 ), z 1 i , x1 ].
Pre-multiplying the original variables by the square root of the variance covariance matrix of it in the 2SLS model renders the HT relatively efficient [START_REF] Hausman | Panel Data and Unobservable Individual Effects[END_REF]Baltagi et al., 2003). [START_REF] Greene | Econometric Analysis, Fifth Edition[END_REF]

Empirical Application and Results

We apply our production modeling framework to a panel of farms in the Limousin region of France, compiled by the regional chamber of agriculture, la Chambre d'Agriculture de la Haute-Vienne. The ministry annually surveyed the production practices of 101 farms between 2007-2009, modeling the survey after the common EU Farm Accountancy Data Network (FADN) survey. We match this production data to a 2009 regional survey of farm wetland area to include total wetland area for each of the farms in our sample [START_REF] Boyard | Les milieux humides agricoles[END_REF]. According to the 2010 agricultural census, the Limousin region counts 14,600 farms and 839,000 hectares of utilized agricultural area [START_REF] Agreste | Le recensement agricole 2010 (Census of Agriculture) en Limousin. Ministère de l'agriculture, de l'agroalimentaire et de la forêt Baltagi[END_REF]. Wetlands represent 4.85% of the total forest and farmland area [START_REF] Chabrol | Inventaire et cartographie des zones humides du Limousin[END_REF]. Depending on the share of wetlands in forested areas, agricultural wetlands total between 40,000 and 58,000 hectares. These wetland areas belong to or overlap the 36 sites of special ecological interest designated according to the EU Habitats and Bird Directives, totaling 101,000 ha, with 43,000 ha of agricultural land (Préfecture de la Région Limousin, 2013). Figure 1 depicts the location of farms and wetland survey sites in the study region.

The farms in our study are majority livestock operations that use agricultural land area primarily for grazing purposes. Crop production is secondary, and mainly intended for supplemental feed. The primary costs posed by wetlands to these producers include reduced grazing area and concomitant increases to animal density. Table 1 summarizes the farm survey variables. Average utilized agricultural area (UAA) is roughly 126 ha, and farms produce an average of 96,613 e in annual value of final product. On average, wetlands cover 13.6% of farm UAA, which is higher than for the region as a whole, and this value ranges for the sample from a low of 0.4% to a high of 58%. Just over half of all UAA is rented and close to 40% of operations responded as family farms. More than half of all operations also receive Less Favorable Area (LFA) designation, mainly due to high altitude (over 600 m above sea level), and close to 40% of farms receive Mountain Area status. These designations qualify farms for additional decoupled subsidy payments. Decoupled subsidy payments average 20,541 e , and also include payments for participation in various AES. However, participation in AES targeted specifically to wetlands is less than ten percent. Coupled subsidy payments average 30,667 e and are mainly tied to arable land area and suckling cow livestock numbers.

Regarding AES payments, the most popular measure in the region is the national basic measure, PHAE22 , also known as the agri-environmental grassland premium. Primarily, PHAE2 requires that grassland areas comprise at least 75% of total utilized agricultural area. The average rate of grassland coverage in Limousin is 84%. Additionally, the farm eligible area must include 20% of protected biodiversity elements (e.g., native vegetation, bird habitat), and maintain an animal grazing density of between 0.4 and 1.4 livestock units per hectare. A livestock unit is equivalent to one dairy cow. PHAE2 also imposes limits to tillage, mineral fertilizer and herbicide use.

The basic measure grassland premium is 76 e /ha for both permanent and temporary grasslands.

Wetlands serve as valuable assets to be eligible for the grassland premium since 1 ha of peat land counts as 20 ha of biodiversity elements, namely permanent grassland, or 100 linear meters of hedges. Hence, with very little wetland area, a farmer may declare a large portion of utilized agricultural land area (temporary, permanent, or wet) as temporary grassland, thus maintaining the option to convert permanent grasslands into temporary grassland area without losing the basic measure payment. In practice, the grassland premium tends to protect a very limited share of wetlands, instead providing payments for whole farm grazing land. There is also recent evidence that the grassland premium has failed to avoid, and in some cases has even encouraged, the conversion of permanent grassland in France [START_REF] Desjeux | Evaluating the impact of rural development measures on nature value indicators at different spatial levels: Application to France and the Netherlands[END_REF].

In addition to the basic measure grassland premium, the Limousin region implemented 20 local AESs during our 2007-2009 study period, each offering 5-year contracts to farmers. Of these, 17 target wetlands, frequently in combination with other areas of ecological interests such as species richness and dry natural grasslands. For the region as a whole, 111 farmers entered this type of scheme.

2,400 ha into these AESs, representing 4% to 6% of total wet farmland area (DRAAF Limousin, 2009). The average per ha annual payment is 200 e , ranging from 34 e to 475 e , with 12 out of 17 schemes paying between 180 e and 280 e . The payment rates depend on the specific measures included in each scheme, as well as the designated area location within the region, and are based on ministry estimates of the additional costs incurred by each agri-environmental practice.

Compared to the grassland premium, these local AESs are offered in designated areas only, and do not necessarily involve the entire farm area of the beneficiary. They require management plans that combine several maintenance and restoration practices, adapted to each enrolled land area. Local AES management plans for wetlands typically include bans on mineral fertilizer and herbicide use, no-tillage requirements, prohibitions on grazing during periods deemed critical for biodiversity, delayed mowing dates to protect wild bird reproduction, specific maintenance practices for ditches and hedges, and the eradication of woody plants to prevent water loss in the soil (Préfecture de la Région Limousin, 2013). Higher payment rates correspond to increased commitments on the part of farmers. Producers may combine the local AESs with the basic measure over different portions of the same farm, but they may not receive payments from both programs for the same land area. While our data set does not include disaggregated information on participation for each of the individual AES measures, we do know the maintained wetland area for each farm. We estimate the costs of maintaining these areas on working land, in terms of lost production value, using the production function in (2). We explore several alternative specifications across FE, RE, and HT estimators. Hausman tests of the RE specifications indicate a preference for the FE estimator, with test statistics generally significant below the 0.05 level. We then use the Hausman pretest procedure suggested by Baltagi et al. (2003) to partition the time-varying (x it ) and time-invariant (z i ) variables into exogenous (x 1 it , z 1 i ) and endogenous (x 2 it , z 2 i ) components.

Based on the pretest results, we treat fertilizer, seed and coupled subsidies as endogenous to the unobserved fixed effects, while the remaining variables satisfy the exogeneity condition (fail to reject at any reasonable significance level) in the HT framework. We note that in our case, the endogenous variables are all time-varying, so that we need only use their mean deviations to serve as instruments. Additionally, to account for the presence of some farms with zero fertilizer, zero pesticide, zero seed expenditures or no purchase of animals to adjust the productive herd (variable named animal expenditure), we include indicators to allow for separate production function intercepts in these cases [START_REF] Battese | A note on the estimation of Cobb-Douglas production functions when some explanatory variables have zero values[END_REF][START_REF] Klemick | Shifting cultivation, forest fallow, and externalities in ecosystem services: Evidence from the Eastern Amazon[END_REF].

We present the results of four specifications in Tables 2 and3, alternately controlling for subsidy payments (coupled and decoupled) and land area designation (mountain area and less favorable area). The coefficient estimates for the production inputs and wetland area variables are generally robust to model specification. While the coefficient estimate magnitudes vary somewhat, the sign and significance levels remain largely the same across models. The coefficient on wetland area is negative and significant across specifications, indicating that a tradeoff between wetland area and production does indeed exist for farms in this region. This is consistent with previous studies that also find that wetland areas pose additional costs to agricultural producers [START_REF] Heimlich | Costs of an Agricultural Wetland Reserve[END_REF][START_REF] Shultz | Implicit Prices of Wetland Easements in Areas of Production Agriculture[END_REF][START_REF] Gelso | Farmers' Perceived Costs of Wetlands: Effects of Wetland Size, Hydration, and Dispersion[END_REF][START_REF] Cortus | The Economics of Wetland Drainage and Retention in Saskatchewan[END_REF]. The significant coefficients on the standard productive inputs are all positive as expected, while land area, fertilizer, seed, and pesticide expenditures are not significant. We note that our sample consists primarily of cattle and dairy farms, which may explain the lack of significance for these standard crop inputs. The coefficient on percent wetland ranges from -0.48 to -0.68, which can be interpreted as the effect of a percentage point increase in wetland area in terms of percentage change in final production value.

Using our most conservative HT estimate, a coefficient of -0.48 implies that a one percentage point increase in wetland area results in a 0.48 percent decrease in final production value. To translate this to monetary terms, we can apply these estimates to average values for UAA and value of final product. This implies that an additional 1.26 ha of wetland area results in a loss of approximately 464 e in final production value. This estimated cost exceeds current AES payments, which are generally between 180 e and 280 e per ha. Moreover, for many farmers, our estimated loss takes into account the grassland premium of 76 e per ha that could not be combined with any of the local AES payments. This poses an additional opportunity cost. For It is important to note that the connection of coupled subsidy payments to production factors, such as arable land area and livestock numbers, likely increases the potential for endogeneity in our model. Unfortunately, while the HT framework allows us to estimate the time-invariant effect of wetland area (our main variable of interest) by instrumenting for endogeneity to the unobserved fixed effect, a i , it does not allow us to simultaneously instrument for endogeneity to the time-varying disturbance, u it . Coupled subsidy payments are tied to production, likely violating exogeneity to u it . To better understand the sensitivity of our results to coupled subsidy payments (separately from decoupled subsidies, which do not depend on production output), we examine an alternate specification of the four models above, omitting coupled subsidy payments entirely, in Tables 4 and5. We find that the same general relationship between wetland area and production value holds, although our upper end HT estimate is now much closer in magnitude to the RE estimates across specifications. The HT wetland area coefficient now ranges from -0.44 to -0.48. An elasticity of -0.44 translates to roughly 425 e for an additional 1.25 ha of wetland area.

Conclusion

Agricultural wetlands are semi-natural habitats [START_REF] Dupraz | Institutional approaches to sustain rural landscapes in France[END_REF]. High ecological and hydrological values of these wetlands are associated with particular practices characterized by the maintenance of permanent grassland with adapted fertilization, grazing, and mowing intensity. Due to lower agricultural productivity, well-functioning wetlands are threatened by either agricultural abandonment, leading to afforestation and drying, or conversion to intensive arable cropping by drainage [START_REF] Russi | The Economics of Ecosystems and Biodiversity for Water and Wetlands[END_REF].

As policy emphasis increasingly shifts from land retirement to conservation practices on working land [START_REF] Baylis | Agri-Environmental policies in the EU and United States: A comparison[END_REF], it is important to understand the associated production effects for determining appropriate AES payment rates and predicting AES participation. We use a production function modeling approach to estimate the cost of maintaining wetland areas on agricultural land, for a panel of livestock producers in the Limousin region of France. We employ a Hausman-Taylor specification to jointly accommodate time-varying and time-constant factors of interest. Our data set includes detailed production information and farm characteristics, similar to the EU FADN, as well as information on farm wetland area. We find that wetland areas on working land do pose significant production costs for farms in our study area, and our results are relatively robust to multiple model specifications. Our most conservative HT elasticity estimate is -0.44, which translates to approximately 425 e in lost production value from an additional 1.25 ha of wetland area. For livestock farms, these costs arise mainly from the loss of productive grazing area and less arable land for producing crops to use as feed.

Our results indicate that the production costs of maintaining wetland areas on working land exceed related AES payments for the majority of producers in our study. This likely contributes to current low participation rates for wetland conservation programs in the region. We qualify these results by noting a few important limitations. First, we work with a relatively small sample of farms and limit our analysis to majority livestock (as opposed to crop) producers. Related to this, our panel covers just three years, during which wetland area on individual farms is constant. Ideally, we would like to model the production effects of changing wetland area over time, especially for understanding the effects of changing participation rates in the AESs. Another limitation concerns the potential for qualitative differences in wetland type. Our survey data does not distinguish between areas that remain saturated year round, versus areas that seasonally fill and drain. Nor does it provide information on the dispersion of wetland area across the agricultural area. These attributes likely affect the cost of a given wetland area [START_REF] Shultz | Implicit Prices of Wetland Easements in Areas of Production Agriculture[END_REF][START_REF] Gelso | Farmers' Perceived Costs of Wetlands: Effects of Wetland Size, Hydration, and Dispersion[END_REF]. That said, by connecting wetland area to detailed production information at the farm level, our data provides a unique opportunity to examine wetland costs directly, at a disaggregated scale.

We have also made a tradeoff here between instrumenting for endogeneity to the unobserved heterogeneity and being able to instrument for endogeneity to any time-varying unobserved factors. Using the HT specification to instrument for the time-invariant effect of wetland area prevents us from instrumenting for endogenous time-varying effects in this case. To mitigate the potential for bias, we include a number of controls for farm characteristics and examine several alternative specifications as a robustness check. We find that our estimates, particularly for wetland effects, are generally robust to model specification. But it is likely that at least some of the production inputs remain endogeneous, particularly variable inputs that are likely chosen with short term production objectives in mind.

Our main results on wetland area are consistent with previous findings that agricultural wetlands do indeed pose significant costs to producers. To our knowledge, this is the first study to apply the production function approach to estimate the cost of maintaining wetlands on working agricultural land. We believe this framework is particularly useful for analysis of AESs, because it allows for direct estimation of participation costs. This is also one of few studies to estimate wetland costs on working land in the EU. Our study contributes to the use of production modeling for estimating AES costs more generally, and we are interested in extending our analysis to better explain participation rates in wetland conservation programs and to modeling the effects of participation on other farm practices, such as the use of chemical inputs or tillage practices.

Les

  Working Papers SMART-LERECO ont pour vocation de diffuser les recherches conduites au sein des unités SMART et LERECO dans une forme préliminaire permettant la discussion et avant publication définitive. Selon les cas, il s'agit de travaux qui ont été acceptés ou ont déjà fait l'objet d'une présentation lors d'une conférence scientifique nationale ou internationale, qui ont été soumis pour publication dans une revue académique à comité de lecture, ou encore qui constituent un chapitre d'ouvrage académique. Bien que non revus par les pairs, chaque working paper a fait l'objet d'une relecture interne par un des scientifiques de SMART ou du LERECO et par l'un des deux éditeurs de la série. Les Working Papers SMART-LERECO n'engagent cependant que leurs auteurs.

Figure 1 :

 1 Figure 1: Farm production and wetland survey areas in the Limousin region

  outlines a feasible weighting procedure using the residuals from the FE model. One practical matter concerns distinguishing the exogenous variables from the endogenous variables. Because exogeneity in this framework surrounds the unobserved fixed effects, we can not directly test for correlation with the observed variables in the model. Instead, we use a Hausman pretest procedure, followingBaltagi et al. (2003), to test for the exogeneity of both the timevarying and time-invariant regressors. This procedure extends the standard use of Hausman testing to choose between FE and RE to the HT estimator. If initial Hausman testing indicates violation of the exogeneity condition for RE, FE is preferred to RE. If Hausman testing on the HT model fails to indicate violation of the exogeneity condition, implying the chosen exogenous regressors have successfully instrumented for the endogenous regressors, HT is preferred to FE.

Table 1 :

 1 Summary statistics

	Variable	Mean Std. Dev. Min	Max
	Value of Final Product	96,613 48,374 2,606 241,208
	Utilized Agricultural Area (Hectares) 126.45	51.19	21.24 342.71
	Labor (hours)	4,267	1,843	2,200 9,240
	Fertilizer Expenditure	7,354	5,222	0.00 27,665
	Seed Expenditure	2,383	2,191	0.00 15,106
	Pesticide Expenditure	1,612	1,852	0.00 11,986
	Feed Expenditure	19,414 14,932	129 168,867
	Fuel expenditures	6,342	3,429	92	20,552
	Animal Expenditure	3,463	8,111	0.00 60,879
	Capital Expenditure	42,160 20,339 2,355 105,914
	No Fertilizer	.035	.184	0.00	1.00
	No Pesticide	.105	.307	0.00	1.00
	No Seed	.058	.234	0.00	1.00
	No Animal Expenditure	.450	.498	0.00	1.00
	Percent Wetland	.136	.122	.004	.583
	Percent Rented Area	.523	.304	0.00	1.00
	Family Farm	.384	.487	0.00	1.00
	Less Favorable Area	.547	.499	0.00	1.00
	Mountain Area	.384	.487	0.00	1.00
	Operator Age	41.54	10.05	22	63
	Subsidies (Coupled)	30,667 18,043	4.82 89,558
	Subsidies (Decoupled)	20,541 15,164	0.00 72,348

Table 4 :

 4 Model Results Part III

		(1)	(2)	(3)	(4)	(5)	(6)
		FE1	RE1	HT1	FE2	RE2	HT2
	Log UAA	0.135	0.0345	0.0431	0.0825	0.0558	0.0618
	Log Labor Hours	0.0602	0.169**	0.166**	0.0908	0.163**	0.164**
	Log Fer Exp	-0.00276	0.0222	-0.00317 -0.00920	0.0193	-0.00643
	Log Seed Exp	-0.0135	0.00971 -0.00126 -0.00875	0.00837	-0.00244
	Log Pesticide Exp	0.0256	0.0266	0.0343*	0.0252	0.0258	0.0342*
	Log Feed Exp	0.210*** 0.273*** 0.267*** 0.221*** 0.278*** 0.275***
	Log Animal Exp	0.0387** 0.0397** 0.0406** 0.0397** 0.0404** 0.0416**
	Log Capital Exp	0.466*** 0.321*** 0.336*** 0.398*** 0.283*** 0.296***
	Log Fuel Exp	0.126	0.119*** 0.124***	0.110	0.108**	0.114**
	No Fertilizer	-0.514*	-0.311	-0.542**	-0.525*	-0.292	-0.516**
	No Seed	-0.292	-0.114	-0.214	-0.227	-0.0868	-0.175
	No Pesticide	0.203	0.187	0.235*	0.210	0.188	0.237*
	No Animal Exp	0.298*	0.282**	0.301**	0.314**	0.300**	0.320**
	Percent Wetland	.	-0.462** -0.480**	.	-0.424** -0.444**
	2008	-0.0725* -0.0570* -0.0673** -0.0649* -0.0554* -0.0653**
	2009	-0.0787** -0.0617* -0.0710** -0.0763** -0.0645** -0.0728**
	Log Subsidies (D)				0.0319*	0.0335** 0.0325**
	Constant	1.694	1.979*** 2.054***	2.163	2.077*** 2.128***
	R 2	0.827	0.857		0.836	0.859

[START_REF] Brander | The Empirics of Wetland Valuation: A Comprehensive Summary and a Meta-Analysis of the Literature[END_REF] conduct a meta-analysis of more than 190 wetland valuation studies, employing a range of valuation methods and with applications to a variety of international settings.

PHAE2 stands for "Prime Herbagère Agro-Environnementale 2" where 2 indicates the second generation of

* p < 0.10, ** p < 0.05, *** p < 0.01

Acknowledgements

The authors wish to thank the regional Chambers of Agriculture of Limousin and Haute-Vienne, for granting us financial support, within the research and development program "Milieux Humides Agricoles" (agricultural wetlands), co-financed by the Ministry of Agriculture, the Limousin Regional Council and the Water Basin Agencies of Loire Bretagne and Adour-Garonne, and for providing us access to the farm level survey data, as well as INRA (French National Institute for Agricultural Research) for co-funding this work. We are also grateful to Yann Desjeux for providing helpful research support.


Emmanuel Paul

Marie-Laure Franco-Montoya

Erwan Paineau

Bernard Angeletti

Shamila Vibhushan

Audrey Ridoux

Arnaud Tiendrebeogo

Murielle Salome

Bernhard Hesse

Delphine Vantelon

Jérôme Rose

Florence Canouï-Poitrine

Jorge Boczkowski

Sophie Lanone

Christophe Delacourt

Jean-Claude Pairon

Pulmonary exposure to metallic nanomaterials during pregnancy irreversibly impairs lung development of the offspring

Keywords: nanoparticles, toxicology, lung development, pregnancy Word count: 6933

Pulmonary exposure to metallic nanomaterials during pregnancy irreversibly impairs lung development of the offspring

Due to the growing commercial applications of manufactured nanoparticles, toxicological studies on nanoparticles, especially during the critical window of development, are of major importance. The aim of the study was to assess the impact of respiratory exposure to metallic and metal oxide nanoparticles during pregnancy on lung development of the offspring and to determine the key parameters involved in lung alterations. Pregnant mice were exposed to weekly doses of 100 µg (total dose 300 µg) of titanium dioxide (TiO 2 ), cerium oxide (CeO 2 ), silver (Ag) nanoparticles or saline solution by nonsurgical intratracheal instillation. The offspring lungs were analyzed at different stages of lung development: fetal stage (gestational day 17.5), pulmonary alveolarization (post-delivery day 14.5), and lung maturity (post-delivery day 49.5).

Regardless of the type of nanoparticle, maternal exposure during gestation induced long-lasting impairment of lung development of the offspring. This effect was accompanied by: i) decreased placental efficiency together with the presence of nanoparticles in placenta, ii) no increase of inflammatory mediators present in amniotic fluid, placenta or offspring lungs, and iii) decreased pulmonary expression of vascular endothelial growth factor α (VEGF-α) and matrix metalloproteinase 9 (MMP-9) at the fetal stage, and fibroblast growth factor 18 (FGF-18) at the alveolarization stage.

Respiratory exposure to metallic nanoparticles during pregnancy induces stereotyped impairment of lung development with a lasting effect in adult mice, independently of the chemical nature of the nanoparticle.

Phone: +33157022093

Email: jc.pairon@chicreteil.fr

Introduction

Nanosciences and nanotechnologies are contributing to new applications in many fields, such as medicine [START_REF] Smith | Selective uptake of single-walled carbon nanotubes by circulating monocytes for enhanced tumour delivery[END_REF][START_REF] Paul | Mn(II)-containing coordination nanoparticles as highly efficient T(1) contrast agents for magnetic resonance imaging[END_REF], antibacterial materials [START_REF] Lu | In situ synthesis of silver nanoparticles uniformly distributed on polydopamine-coated silk fibers for antibacterial application[END_REF], foods [START_REF] Martirosyan | Engineered nanomaterials in food: implications for food safety and consumer health[END_REF], cosmetics [START_REF] Bowman | Nanomaterials and regulation of cosmetics[END_REF], pigments [START_REF] Shi | Titanium dioxide nanoparticles: a review of current toxicological data[END_REF], and electronics [START_REF] Shen | Preparation of solid silver nanoparticles for inkjet printed flexible electronics with high conductivity[END_REF]. Titanium dioxide (TiO 2 ), cerium oxide (CeO 2 ), and silver (Ag) nanoparticles (NPs) are among the most commonly manufactured NPs over the last decade. Pulmonary exposure of humans to these latter NPs is rapidly becoming unavoidable, raising several questions with regard to their safety and environmental impact. Experimentally, in vivo administration of CeO 2 NPs or Ag NPs into the airways of adult animals induces toxicological effects, such as oxidative stress [START_REF] Srinivas | Acute inhalation toxicity of cerium oxide nanoparticles in rats[END_REF], inflammation [START_REF] Haberl | Cytotoxic and proinflammatory effects of PVP-coated silver nanoparticles after intratracheal instillation in rats[END_REF], or lung remodeling [START_REF] Ma | Induction of pulmonary fibrosis by cerium oxide nanoparticles[END_REF].

The potential toxicological effects of pulmonary exposure to NPs during pregnancy, which is a critical window of development, has been poorly documented. Previous experimental studies have strongly suggested that NPs might impair fetal development, although most of these results were obtained using exposure conditions that were not relevant to usual human environmental exposure [START_REF] Shimizu | Maternal exposure to nanoparticulate titanium dioxide during the prenatal period alters gene expression related to brain development in the mouse[END_REF][START_REF] Philbrook | The effect of TiO(2) and Ag nanoparticles on reproduction and development of Drosophila melanogaster and CD-1 mice[END_REF][START_REF] Yamashita | Silica and titanium dioxide nanoparticles cause pregnancy complications in mice[END_REF]. Interestingly, the study by Hougaard et al. showed that inhalation of TiO 2 NPs during pregnancy induced moderate neurobehavioral changes in the offspring [START_REF] Hougaard | Effects of prenatal exposure to surface-coated nanosized titanium dioxide (UV-Titan). A study in mice[END_REF]. NPs can cross the placental barrier in pregnant mice after intravenous [START_REF] Yamashita | Silica and titanium dioxide nanoparticles cause pregnancy complications in mice[END_REF] or oral exposure [START_REF] Philbrook | The effect of TiO(2) and Ag nanoparticles on reproduction and development of Drosophila melanogaster and CD-1 mice[END_REF] with demonstration of the presence of NPs in fetuses using TEM, and this translocation was linked to an impairment of normal growth and development of the fetuses. To our knowledge, no study has evaluated the impact of prenatal exposure to NPs on lung development. The aim of the present study was to evaluate the impact of exposure of pregnant mice to various intratracheally instilled NPs on the lung development of their offspring. Because NPs physicochemical characteristics are important determinants of their toxicological effects [START_REF] Oberdorster | Safety assessment for nanotechnology and nanomedicine: concepts of nanotoxicology[END_REF], we used NPs with similar shapes and sizes, but with different physicochemical properties: TiO 2 NPs which are poorly soluble in biological media, CeO 2 NPs which have a high standard reduction potential (i.e. strong oxidant) and Ag NPs which dissolve through Ag oxidation. Our results show that maternal pulmonary exposure of mice to various NPs induced impairment of lung development with lasting effects in adult animals, regardless of the metallic or metal oxide NPs tested and regardless of the pulmonary response observed in the dams.

Methods

Nanoparticle preparation and characterization

TiO 2 , CeO 2 and polyvinylpyrrolidone (PVP)-coated Ag nanopowders were purchased from Nanostructured and Amorphous Materials, Inc. (Houston, USA), Umicore, Nanograin ® (Olen, Belgium) and nanoComposix, Inc. (Prague, Czech Republic), respectively. All nanoparticles were sold as having a spherical shape, 10 nm in diameter.

The various NP powders were suspended in 0.9% NaCl at a concentration of 10 mg/ml. Before administration, NP suspensions were vortexed and sonicated in an ice bath at 37 kHz for 10 minutes. The size distribution was assessed by transmission electron microscopy (TEM, JEOL JEM 1400, Peabody, USA) based on examination of more than 1,500 particles. Dynamic light scattering (DLS, NanoZS, Malvern, UK) was carried out to determine the zeta potential and hydrodynamic diameter of the various NPs. The crystallinity of TiO 2 NPs and CeO 2 NPs was determined by X-ray diffraction (XRD, PANalytical X'Pert PRO, Limeil-Brévannes, France). The specific surface area of TiO 2 NPs was measured by a Micromeritics Flowsorb 2300 (Norcross, GA) using Brunauer Emmett Teller (BET) analysis.

The specific surface area of CeO 2 NPs and Ag NPs was estimated by TEM. To investigate possible Ag ion release from the Ag NPs suspension, an additional experiment was performed using Inductively Coupled Plasma Mass Spectrometry (ICP-MS, Perkin Elmer NexIon 300X, Waltham, USA). NPs endotoxin content was examined using the Limulus Amebocyte Lysate (LAL) kit QCL-1000 (Lonza, Basel, Switzerland) according to the manufacturer's instructions.

Animals and treatments

Twelve-week-old C57BL6/J mice (88 multiparous females and 44 males) were purchased from Janvier Laboratories (Le Genest-Saint-Isle, France). Multiparous females (with only 1 previous birth) were used in view of the high risk of cannibalism or abandonment by primiparous dams, as previously observed in the literature [START_REF] Weber | Influence of strain and parity on the risk of litter loss in laboratory mice[END_REF]. Male and female mice were housed separately under controlled room temperatures (22 ± 2 °C), a 12hour dark/light cycle with ad libitum access to food and water. Ethical approval for all animal experimentation was obtained from the local ethics committee (N° 12-104, ComEth Afssa/ENVA/UPEC, Maisons-Alfort, France). After one week of acclimatization, females were placed with males overnight (12 h). The following morning, mating was confirmed by the presence of a vaginal plug, and this time-point was considered to be gestational day 0.5 (GD 0.5). Pregnant mice were randomly divided into four groups (n ≥ 20 per group): saline, TiO2 NPs, CeO2 NPs, and Ag NPs. Dams were housed 2 per cage, and cages had sawdust (changed weekly), cotton for nests, and a piece of wood to play. All procedures were conducted in accordance with the ethical standards of the local committee on animal experiments. At the time of exposure, pregnant mice were anesthetized by intramuscular injection (150 µl per mouse) of a mixture of 10% ketamine (Merial, Lyon, France) and 10% xylazine (Bayer, Puteaux, France) in physiological saline. For the NP-exposed groups, 10 µl of NPs suspended at 10 mg/ml were administered by non-surgical intratracheal instillation, three times during gestation (once a week: on GD 2.5, GD 9.5 and GD 16.5) using a 22 gauge catheter, while 10 µl of vehicle alone was injected for the saline group. The total dose is 300 µg, in the same order of magnitude as the dose previously used by Hougaard et al. [START_REF] Hougaard | Effects of prenatal exposure to surface-coated nanosized titanium dioxide (UV-Titan). A study in mice[END_REF]. Pups were examined at various time-points during development, two days prior to spontaneous delivery (GD 17.5) for fetotoxicity analysis and measurement of metal concentrations in tissues, during lung alveolarization on post-delivery day 14.5 (PD 14.5) and when mice were adults (PD 49.5) for lung morphometry. The experimental protocol design is presented in supplementary Figure S1.

Fetotoxicity

To evaluate the potential fetotoxicity induced by NPs exposure, a C-section (n ≥ 6 /group) was performed in dams at GD 17.5 and followed by rapid decapitation of fetuses.

Uterine weight, number of fetuses, fetal weight (n ≥ 37/group), fetal resorption (number of resorptions/total number of formed fetuses and resorptions) and placental efficiency (fetal weight/placental weight) were scored.

Cytokine and chemokine determination in amniotic fluid and fetal lungs

To further elucidate the toxicological mechanisms induced in pups after NP exposure during pregnancy, cytokine and chemokine concentrations were measured using the Luminex assay in the amniotic fluid of each pregnant dam and in the fetal lungs. The uterus was removed at GD 17.5 and 50 µl of amniotic fluid were collected by direct insertion of a fine needle into the amniotic cavity. Immediately after harvesting, amniotic fluids were centrifuged (8 min at 10,000 rpm) and the supernatants were stored at -80 °C. Fetal lungs were homogenized in 500 µl of RIPA (Sigma-Aldrich, Saint-Louis, USA) lysis buffer with 10% of protease inhibitor cocktail (Sigma-Aldrich, Saint-Louis, USA) and stored at -80 °C.

To evaluate inflammatory response or immune response, 26 cytokines or chemokines were measured in amniotic fluid (n= 19/group) and fetal lungs (n= 10/group) in a total volume of 50 µl using the Luminex assay (MCYTOMAG-70K-26, Merck Millipore, Saint-Quentin en Yvelines, France).

Chemical analysis of placenta and fetal lungs

In order to determine whether NPs can cross the placental barrier and have a direct effect in the developing fetus, chemical analysis was performed using ICP-MS. At GD 17.5, pregnant mice (n ≥ 6/group) were sacrificed after being anesthetized, and the placenta (n ≥ 10/group) and fetal lung (n ≥ 16/group) were collected and stored at -20 °C for ICP-MS analysis. Two placentas and 4 fetal lungs from each exposure group were pooled to determine dry organ weights. All tissues were digested with 1 ml of 67-69% nitric acid (Plasmapure Plus SCP science, Courtaboeuf, France) for 1 hour at room temperature. During the digestion process, 0.5 ml of 30% hydrogen peroxide (Plasmapure Plus SCP science), 1 ml of 40% hydrofluoric acid (Plasmapure Plus SCP science) and 100 µl of 96% sulfuric acid (Plasmapure Plus SCP science) were added to each sample. Samples were heated for 1.5 hour in a microwave chamber (UltraWAVE Milestone, Sorisole, Italy). Metal concentrations, expressed in µg/g for each sample, were measured by ICP-MS.

The localization and speciation of Ag in the placenta and fetal lungs were assessed using micro X-ray fluorescence (micro-XRF) and micro X-ray absorption near edge structure (micro-XANES). Prior to measurements, tissues were fixed in 10% formalin overnight, dehydrated in a graded series of ethanol and isopropanol solution, and embedded in paraffin.

Tissue sections (15 µm thick) were cut with a microtome and placed on thin window films used for XRF studies (Ultralene ® , 4 µm thick, SPEX SamplePrep).

XRF microscopy experiments were performed on ID21 beamline of the European Synchrotron Radiation Facility (ESRF, Grenoble, France) [START_REF] Szlachetko | Wavelength-dispersive spectrometer for X-ray microfluorescence analysis at the X-ray Microscopy beamline ID21 (ESRF)[END_REF]. The X-ray beam was monochromated by a fixed-exit double-crystal Si(111) monochromator (Kohzu, Japan) and beam focalization was ensured by Kirkpatrick Baez (KB) mirrors allowing a beam size of 0.4*0.9 µm². Incident X-ray energy of 6.7 keV was chosen in order to excite the Ag Llines (L α = 2.98 and L β = 3.15 keV). A silicon drift diode detector (SDD, Bruker) was used for fluorescence detection. Raster scans were performed across a chosen area of the sample preparation. Analysis of the X-ray fluorescence spectrum for each pixel can be used to plot a separate chemical map for each element. Analysis of XRF data was performed by batch processing using PyMca software (V.A. [START_REF] Solé | A multiplatform code for the analysis of energy-dispersive X-ray fluorescence spectra[END_REF].

XANES measurements were performed to determine the speciation of Ag NPs in the placenta. X-ray absorption spectra at the AgL 3 edge (3.351 keV) were collected at the LUCIA beamline of the SOLEIL synchrotron (Orsay, France) [START_REF] Vantelon | The LUCIA beamline at SOLEIL[END_REF]. The X-ray beam was monochromated with a Si(111) crystal and beam focalization was ensured by Kirkpatrick Baez (KB) mirrors, allowing a beam size of 3*3 µm² on the sample. XANES data were obtained after performing standard procedures for pre-edge subtraction and normalization using IFEFFIT implemented in the ATHENA software package [START_REF] Ravel | HEPHAESTUS: data analysis for X-ray absorption spectroscopy using IFEFFIT[END_REF].

Lung morphometry

Lung histology was analyzed in mothers and lung morphometry was measured in pups. Lungs were inflated with 10% prefiltered formalin at a pressure of 20 cmH 2 O using a 22 gauge catheter, and were fixed overnight at room temperature. Lung volume was determined using the fluid displacement method [START_REF] Franco | LPS-induced lung injury in neonatal rats: changes in gelatinase activities and consequences on lung growth[END_REF]. After fixation, tissues were dehydrated in a graded series of ethanol and isopropanol solution and embedded in paraffin.

Five micrometer thick medial frontal sections of lungs were stained with hematoxylin and eosin (H&E). An experimental pathologist reviewed all lung slides blindly with respect to the animal's treatment, and semi-quantitative evaluation of the inflammatory infiltrate (based on the presence of neutrophils and macrophages) was performed. Airway wall thickness was measured using imageJ software (National Institute of Health, USA) for evaluation of bronchial changes after intratracheal instillation of NPs in the lungs of the dams (n≥4/group).

The mean of 5 measures per complete bronchus was calculated, and 10 randomly selected bronchi per lung were evaluated. To assess the histopathological changes in pups, offspring were killed at PD 14.5 and PD 49.5 by exsanguination after aortic section. Lung sections of 10 pups randomly selected from each group were analyzed at the two different time points PD 14.5 and PD 49.5 using light microscopy (Axioplan 2, Oberkochen, Germany). Twenty randomly selected fields for each mouse (10 per lung) were studied at x10 magnification.

Lung alveolarization defects were measured using two methods: mean linear intercept (MLI) and radial alveolar count (RAC). MLI was quantified by the methods described by Weibel [START_REF] Weibel | Morphometric studies on the growth of gas exchange capacity of the rat lung[END_REF] and RAC was determined as previously reported [START_REF] Cooney | The radial alveolar count method of Emery and Mithal: a reappraisal 2--intrauterine and early postnatal lung growth[END_REF]Thurlbeck, 1982, Emery and[START_REF] Emery | The number of alveoli in the terminal respiratory unit of man during late intrauterine life and childhood[END_REF].

Genes and proteins expression in placenta and pup's lungs

mRNA was isolated from whole-lung tissue homogenates using the RNeasy kit (Qiagen, Venlo, The Netherlands). Reverse transcription reactions were performed with Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV, Life Technologies) according to the manufacturer's instructions. The expression of genes involved in inflammatory response in the placenta and in lung development (Table S1, supplementary data) was determined by RT-PCR using the Sybr Select Master Mix (Life Technologies) in a QuantStudio 6 Flex Real-Time PCR System (Life technologies). mRNA expression was normalized to that of HPRT1 and 18S as a housekeeping gene. The sequences of the primers used are shown in Table S1 (supplementary data). Placental genes expression was performed in two layers of placenta, spongiotrophoblast and labyrinth zone.

Pup's lungs were homogenized in 1 ml of RIPA (Sigma-Aldrich, Saint-Louis, USA) lysis buffer with 10% protease inhibitor cocktail (Sigma-Aldrich, Saint-Louis, USA) and stored at -80 °C. MMP-9 and VEGF levels in lung homogenate were detected by ELISA according to the manufacturer's instructions and the optical density was determined at 450 nm with a microplate reader (Infinite® 200 Pro, Tecan, Männedorf, Switzerland).

Statistics

Results of NP characterization were expressed as mean ± standard error of the mean (s.e.m), while other results were expressed as median and interquartile due to non-normal distribution. As the data present a hierarchical structure (several offspring/placenta or several fetuses per dam) leading to a possible litter effect, we analyzed the relationship between exposure groups and fetal weight, placental weight, placental efficiency, cytokine and chemokine levels in amniotic fluid and fetal lungs, chemical assays in placenta and fetal lungs, XANES measurement and lung morphometry measurements and gene and protein expression in pup lungs using a mixed regression model (separately for each measure). The saline group constitued the reference group. Log-transformation was performed to normalize the distribution of biomarkers. The absence of any significant difference between the mixed model and the linear regression model (likelihood ratio test) indicated the absence of significant intra-class correlation (i.e litter effect), and the more parsimonious model (linear regression model) was used. The gender effect was tested by adjusting for gender in the regression model of lung morphometry measurements (at PD 14.5 and 49.5). All tests were two-tailed. A p-value less than 0.05 was considered statistically significant. Data were analyzed with GraphPad Prism 6.0 (La Jolla, CA) and STATA v13.0 (College Station, TX, USA). p-value less than 0.05 was considered statistically significant. Data were analyzed with GraphPad Prism 6.0 (La Jolla, CA) and STATA v13.0 (College Station, TX, USA).

Results

Nanoparticle characterization

Transmission electron microscopy (TEM) images showed that all three NPs, i.e. TiO 2 NPs, CeO 2 NPs and Ag NPs, were spherical and had a similar primary particle size of 12.30.1, 22.40.2, and 10.40.1 nm, respectively (Supplementary Figure S2). DLS analysis after suspending NPs in NaCl at a concentration of 10 mg/mL, indicated an aggregation state for TiO 2 and CeO 2 NPs that was higher (with aggregate size > 1000 nm) than that for Ag NPs (with aggregate size < 1000 nm) (Supplementary Figure S3). NPs characteristics are summarized in Table 1. Silver ion dissolution from the Ag NPs suspension represented less than 3% of total silver in the Ag NPs suspension, which suggested that the Ag NPs instilled into pregnant mice were in nanoparticle form.

Fetotoxicity of nanoparticles

Results of fetotoxicity analysis at gestational day 17.5 (GD 17.5) are shown in Figure 1. Uterine weight was not significantly modified after NPs treatment (Figure 1B). When compared to the control group, mice that received Ag NPs had significantly higher fetal resorption rates (p=0.004), while TiO 2 NPs and CeO 2 NPs treatment did not affect fetal resorption rates (Figure 1C,D). The number of living fetuses per litter was decreased in Ag groups (Figure 1E), and fetal weight (GD 17.5) was significantly decreased in the 3 NPexposed groups compared to the control group (p<0.05). This decrease was much more marked for the Ag NPs group (p<0.001) (Figure 1F,G). Moreover, placental weight of CeO 2 and Ag NPs groups was significantly higher than in the control group (Supplementary Figure S4). This was accompanied by a decrease of placental efficiency for all NP-exposed groups compared to the control group (Figure 1H).

Lung morphometric measurement

Light microscopy evaluation of pup's lungs showed that pulmonary exposure to the three NPs during pregnancy induced significant and similar increases in the mean linear intercept (MLI) and decreases in the radial alveolar count (RAC) (Figure 2) and lung alveolar surface as compared to control animals (Table 2). Pulmonary exposure during pregnancy induced an increase of the alveolar space and a decrease of the number of alveoli, therefore globally leading to enlargement of the alveolar space. These phenomena were observed at the two stages of lung development studied (PD 14.5 and PD 49.5).

Effects of NPs on pregnancy and dam lungs

Maternal body weight of TiO 2 NPs-and CeO 2 NPs-treated mice did not differ from that of the saline group, and was slightly, but significantly decreased in Ag NPs-treated mice at GD 17.5 (Figure 1A). The number of inflammatory cells, such as macrophages and neutrophils, was increased in the lungs of pregnant mice after all NPs treatment compared to the control group. Dense areas observed on histological sections of dam lungs can be attributed to TiO 2 NPs and CeO 2 NPs aggregation (indicated as arrows). Such dense zones were present in the alveolar space, and were also seen in macrophages (Supplementary Figure S5A). Small airway wall thickness was measured as index of bronchial alteration and the results showed an increase of wall thickness in the lungs of pregnant mice after NPs exposure, which was significant in the CeO 2 NPs and Ag NPs groups compared to the saline group (Supplementary Figure S5B).

Cytokine content in amniotic fluid and fetal lungs

Among the various cytokines and chemokines analyzed in amniotic fluid, the expression of vascular endothelial growth factor (VEGF) and macrophage inflammatory protein 2 (MIP-2), were significantly decreased in CeO 2 and Ag NPs-exposed groups as compared to the control group (Supplementary Table S2). In addition, Granulocytemacrophage colony-stimulating factor (GM-CSF), interleukin 10 (IL-10) and interleukin 15 (IL-15) levels were decreased in the Ag NPs-exposed group as compared to the control group.

The expression of monocyte chemoattractant protein-1 (MCP-1) significantly decreased in the TiO 2 NPs-exposed group as compared to the control group. Only the expression of interferon gamma-induced protein 10 (IP-10) cytokine was increased in amniotic fluid in the Ag NPsexposed group as compared to the control group. No difference was observed for the other cytokines and chemokines examined. In fetal lung, cytokine and chemokine levels were similar for almost of them in the 3 NPs groups as compared to the saline group (Supplementary Table S3). GM-CSF protein expression was significantly decreased in the CeO2 NPs and Ag NPs-exposed groups. VEGF and IP-10 proteins expression was also significantly decreased in the Ag NPs group as compared to the control group. Conversely, granulocyte-colony stimulating factor (G-CSF) and IL-10 were significantly increased in the TiO 2 NPs-exposed groups as compared to the control group. Moreover, IL-12 (p40) protein level was significantly increased in the CeO 2 NPs-exposed group as compared to the control group.

Biodistribution of nanoparticles and metal burden in placenta and fetal lung

In accordance with exposure, titanium (Ti), cerium (Ce) and silver (Ag) burdens determined by ICP-MS were significantly higher in the placentas of the respective NPstreated groups as compared to the control group (Table 3). Analysis of metal burden levels in fetal lung at GD 17.5 revealed significant accumulation only for Ag (Table 3). To more precisely localize Ag NPs, X-Ray fluorescence (XRF) analysis was performed on placenta and fetal lungs of the Ag NPs group (Figure 3). Micro-XANES analysis performed on the same areas showed that Ag could be found in its nano form on the fetal side of the placenta, more particularly as large deposits on the chorionic plate. Ag was also localized as spots with random distribution in fetal lung.

Expression of genes and proteins involved in lung development at GD 17.5 and PD 14.5

Gene expression was measured in pup's lungs in order to elucidate the mechanisms involved in the alteration of lung development. During the fetal stage (GD 17.5), VEGF-α mRNA expression was significantly decreased (p<0.05) in TiO 2 NPs-and CeO 2 NPs-exposed groups as compared to the control group (Figure 4A), whereas MMP-9 mRNA expression was decreased (p<0.05) in all 3 NPs-exposed groups (Figure 4B). At the alveolarization stage (PD 14.5), FGF-18 mRNA expression was significantly decreased (p<0.05) in the 3 NPstreated groups as compared to the control group (Figure S6A). Expression of other genes involved in alveolarization, such as FGF-R3, FGF-R4 and adrenomedullin, was not modified by NPs exposure (Figure S6 B, C andD). VEGF protein expression in pup lungs at PD 14.5 was significantly decreased in the CeO 2 NPs-and Ag NPs-exposed groups as compared to the control group (Figure 4C). MMP-9 expression was similar in the 3 NPs-exposed groups and in the control group (Figure 4D).

Discussion

The main results of this study are that pulmonary exposure during pregnancy to metallic and metal oxide NPs (TiO 2 , CeO 2 and Ag) with similar shapes and primary particle diameters, but with different solubility and surface properties, led to a similar and long-lasting impairment of lung development of the offspring. This effect was associated with: i) a significant decrease in fetal weight and placental efficiency index associated with the presence of the three NPs in the placenta, ii) the absence of any significant amniotic or fetal lung inflammation, and iii) a significant decrease in VEGF mRNA and protein levels in both amniotic fluid and pup lungs. Overall, to the best of our knowledge, these results provide the first experimental evidence of impairment of offspring pulmonary development after Different mechanisms by which pulmonary exposure to NPs during pregnancy could induce lung impairment in the offspring were investigated in the present study. First, we hypothesized that maternal exposure to NP may induce pulmonary inflammation, resulting in a fetal inflammatory condition and delayed lung development, as previously reported [START_REF] Kramer | Prenatal inflammation and lung development[END_REF]. Histological examination of the lungs of exposed pregnant mice demonstrated that NP exposure induced pulmonary inflammation in the 3 groups of NP-treated animals. This type of pulmonary inflammation response to respiratory exposure to NP has been previously reported in the literature [START_REF] Seiffert | Pulmonary toxicity of instilled silver nanoparticles: influence of size, coating and rat strain[END_REF][START_REF] Srinivas | Acute inhalation toxicity of cerium oxide nanoparticles in rats[END_REF][START_REF] Hougaard | Effects of prenatal exposure to surface-coated nanosized titanium dioxide (UV-Titan). A study in mice[END_REF]. However, in our study, no inflammatory response was detected in the amniotic fluid, placenta (Figure S7, supplementary data) and lungs of fetuses from dams exposed to NPs. In particular, no increase in IL-6 and TNF-α levels was observed in amniotic fluid. This could be considered as striking as such increase has been described as predictive of fetal inflammatory response and adverse neonatal outcome [START_REF] Kunze | Cytokines in noninvasively obtained amniotic fluid as predictors of fetal inflammatory response syndrome[END_REF]. However, this particular study was performed in a very different context (i.e. human with fetal inflammatory response syndrome, due to intrauterine inflammation and/or infection), which could largely influence the results. In some experimental studies, a significant increase in IL-6 and TNF-α levels was described. However, this was observed in adults mice lungs only after pulmonary exposure to high doses of particles (TiO 2 , diesel exhaust) or very inflammogenic particles (Quartz) [START_REF] Saber | Cytokine expression in mice exposed to diesel exhaust particles by inhalation. Role of tumor necrosis factor[END_REF][START_REF] Roursgaard | Time-response relationship of nano and micro particle induced lung inflammation. Quartz as reference compound[END_REF][START_REF] Roursgaard | Acute and subchronic airway inflammation after intratracheal instillation of quartz and titanium dioxide agglomerates in mice[END_REF]. Finally, although the mother's lungs appear as inflammatory, this compartment is far from the intrauterine environment. This, therefore, may explain the lack of increase of IL-6 and TNF-α levels that we observed in the fetal compartment. Inflammation of dam lungs exposed to NPs therefore does not appear to be the underlying mechanism contributing to lung impairment in the offspring.

Second, placental insufficiency is known to be strongly associated with fetal growth restriction (FGR), fetal death and inhibition of fetal development (William W. [START_REF] Hay | Intrauterine growth restriction[END_REF][START_REF] Rosenberg | The IUGR newborn[END_REF]. Moreover, it has been shown that FGR has long-term effects on postnatal lung structure [START_REF] Maritz | Fetal growth restriction has long-term effects on postnatal lung structure in sheep[END_REF]. In our study, we showed that exposure to NPs during pregnancy decreased placental efficiency, leading to fetal death and FGR. FGR could be directly related to translocation of NPs from dam lungs to pups. Fetal death and FGR have been demonstrated after intravenous administration of high doses of NP in pregnant mice [START_REF] Yamashita | Silica and titanium dioxide nanoparticles cause pregnancy complications in mice[END_REF]. In our study, ICP-MS and XRD analysis showed the presence of Ti, Ce and Ag elements in the placenta after exposure to NPs during pregnancy. Moreover, XANES analysis revealed that silver was present in the placenta in its nano form. In view of the lower solubility of TiO 2 NPs and CeO 2 NPs, we can therefore assume that the 3 NPs used in this study were present in their nanoparticle form in the placenta, and could be involved in inflammation-independent decreased placental efficiency with ensuing FGR and impairment of pup lung development. Ag element was also identified in the fetal lung, suggesting that Ag may cross the placental barrier and may have a direct effect on the developing fetus, although the presence of the nanoparticle form of Ag in fetal lung cannot be formally confirmed. Ag element did not appear to be distributed equally in the fetal organs, as Ag was mainly present in the liver and kidneys of the fetuses. Moreover, Ag was also present at a significant degree in the fetal brain (Supplementary Table S4). The form of Ag (nano or ionic) can modify its biodistribution in the organs, as the study by Charehsaz et al. showed that Ag biodistribution in the fetus, after oral gavage during pregnancy of different forms of Ag (nano or ionic), differed according to the two Ag species. Ag element from the AgNO 3 group was detected in the lungs and plasma of the fetuses, whereas in the Ag NPs group, elevated levels of Ag were only observed in the fetal kidney [START_REF] Charehsaz | Effects of developmental exposure to silver in ionic and nanoparticle form: A study in rats[END_REF]. Differences in the biodistribution of the 3 NPs could be explained by the fact that TiO 2 and CeO 2 NPs had a higher aggregation state than Ag NPs, as shown in TEM images and confirmed by DLS analysis. The capacity of NPs to cross the placenta has been previously reported via other routes of administration [START_REF] Yamashita | Silica and titanium dioxide nanoparticles cause pregnancy complications in mice[END_REF][START_REF] Austin | Distribution and accumulation of 10 nm silver nanoparticles in maternal tissues and visceral yolk sac of pregnant mice, and a potential effect on embryo growth[END_REF]. However, the hypothesis of a direct effect of NPs on lung development does not appear to be relevant for all types of NPs because, in the other two exposure groups, no excess of Ti and Ce elements was detected in fetal lung although the degree of lung impairment was similar to that observed in the Ag NPs-treated group. Therefore, our main hypothesis is that administration of NPs in pregnant mice is followed by an effect on the placenta with a subsequent effect on the respiratory development of the offspring, independently of the size of aggregates observed with NPs.

Various signaling pathways may be involved in the alteration of pup lungs development secondary to placenta insufficiency in the present study. We first focused on VEGF, which is involved in microvascular lung development [START_REF] Mullassery | Lung development[END_REF].

The results showed that VEGF expression was significantly decreased in amniotic fluid and also in pup lungs at GD 17.5 and PD 14.5 after treatment of dams with the three NPs. We also observed decreased VEGF gene expression in the placenta, more particularly in the spongiotrophoblast layer for TiO 2 and CeO 2 NPs (Figure S7, supplementary data). This result is in accordance with the recent study by Valentino et al., who observed a decrease in placental blood flow following maternal exposure to diluted diesel engine in a rabbit model [START_REF] Valentino | Maternal exposure to diluted diesel engine exhaust alters placental function and induces intergenerational effects in rabbits[END_REF] but also with other studies in mice [START_REF] Yamashita | Silica and titanium dioxide nanoparticles cause pregnancy complications in mice[END_REF][START_REF] Qi | Damaging effects of multiwalled carbon nanotubes on pregnant mice with different pregnancy times[END_REF]. Decreased placental blood flow may affect maternal-fetal exchange and consequently fetal growth. In this context, the decreased VEGF observed in pup lungs could be due to the presence of NPs in this organ. However, only the presence of Ag NPs was observed in pup lungs, while similar degrees of decreased VEGF and lung impairment were observed in the 3 groups of pups born to dams exposed to NPs. Decreased VEGF expression could be the mechanism leading to impairment of the lungs, as it has been demonstrated that an inhibitor of the VEGF receptor KDR/flk-1 was associated with hypoalveolarization [START_REF] Jakkula | Inhibition of angiogenesis decreases alveolarization in the developing rat lung[END_REF]. Moreover, inhibition of VEGF signaling after intra-amniotic treatment with Flt-1 may disrupt lung growth and contribute to an increased risk of bronchopulmonary dysplasia [START_REF] Tang | Excess soluble vascular endothelial growth factor receptor-1 in amniotic fluid impairs lung growth in rats: linking preeclampsia with bronchopulmonary dysplasia[END_REF]. Analysis of other genes involved in lung development showed that MMP-9 and FGF-18 were similarly downregulated in the 3 groups of pups. These findings are in accordance with those of a study that investigated pulmonary exposure of newborn mice to TiO 2 NPs, which showed decreased expression of VEGF-α [START_REF] Ambalavanan | Titanium oxide nanoparticle instillation induces inflammation and inhibits lung development in mice[END_REF]. In contrast, the present study provides the first evidence of decreased FGF-18 mRNA expression in mice exposed to NPs during gestation. We also observed decreased expression of MMP-9, while Ambalavanan et al. reported increased MMP-9 expression in newborn mice exposed to TiO 2 NP [START_REF] Ambalavanan | Titanium oxide nanoparticle instillation induces inflammation and inhibits lung development in mice[END_REF]. However, in their study, increased MMP-9 expression was observed in the context of a pro-inflammatory response that was not observed in the present study.

Certain methodological aspects of our study need to be discussed. Firstly, animals were exposed to NP via intratracheal instillation instead of aerosol exposure, which is more relevant in terms of human exposure to manufactured NP. However, intratracheal instillation has been reported to provide similar results in terms of pulmonary exposure to nanomaterials compared to aerosol exposure and similar results in terms of inflammatory respiratory response [START_REF] Morimoto | Comparison of pulmonary inflammatory responses following intratracheal instillation and inhalation of nanoparticles[END_REF]. Mice were exposed weekly to different NPs to ensure exposure of fetuses throughout gestation. We therefore cannot evaluate specific phases of gestation. Another limitation of this study could be the use of a single high dose of NP. We chose this strategy because this study was designed to demonstrate the concept of deleterious effects in the offspring following maternal lung exposure to NP. Nevertheless, the dose chosen was of the same order of magnitude as the dose previously used by Hougaard et al. [START_REF] Hougaard | Effects of prenatal exposure to surface-coated nanosized titanium dioxide (UV-Titan). A study in mice[END_REF] and was also relevant to occupational exposure to metallic NP reported in some occupational settings [START_REF] Lee | Exposure assessment of workplaces manufacturing nanosized TiO2 and silver[END_REF]. Indeed, based on a rough estimate of an equivalent of inhaled aerosol applying the equation proposed by Présumé et al. [START_REF] Presume | Exposure to metal oxide nanoparticles administered at occupationally relevant doses induces pulmonary effects in mice[END_REF], the equivalent inhaled concentration received by the mice in our study was 2.8 mg/m 3 for an exposure of 8h/day for 5 days. Finally, we performed complete physicochemical characterization of the 3 three NPs and analyzed several stages of lung development (fetal stage, alveolarization stage and adult lung), to examine persistence of the effects of in utero exposure to NPs. In humans, it has been demonstrated that children with bronchopulmonary dysplasia may be at increased risk of lung diseases such as emphysema, asthma and Chronic Obstructive Pulmonary Disease-like phenotype later in life (Wong et al., 2008, Baraldi and[START_REF] Baraldi | Chronic lung disease after premature birth[END_REF]. These findings also raise the question of whether, like in utero exposure to cigarette smoke [START_REF] Wu | Prenatal and early, but not late, postnatal exposure of mice to sidestream tobacco smoke increases airway hyperresponsiveness later in life[END_REF][START_REF] Hollams | Persistent effects of maternal smoking during pregnancy on lung function and asthma in adolescents[END_REF][START_REF] Drummond | Combined Effects of in Utero and Adolescent Tobacco Smoke Exposure on Lung Function in C57Bl/6J Mice[END_REF], maternal exposure to NPs during pregnancy could increase the risk of developing lung diseases such as asthma or COPD in adulthood. The clinical relevance of our findings may therefore extend beyond the scope of histological abnormalities and indicate an increased susceptibility to develop other respiratory tract diseases. Specific complementary experiments should be conducted in this context.

Conclusion

In conclusion, the present study provides the first evidence that respiratory exposure during pregnancy leads to long-lasting lung impairment in the offspring. In view of the growing use of these nanoparticles, this result raises concerns for public health and pregnant women and their developing fetus particularly for those at high risk of occupational or domestic exposure.

Tables Table 1:

  respiratory exposure to NPs during gestation. The underlying mechanisms probably involve placental insufficiency secondary to the presence of NPs in this organ with ensuing down regulation of critical mediators of lung development without any amniotic fluid or fetal lung inflammation.

Figure 1 :

 1 Figure 1: Pregnancy complications after NPs treatmentPregnant mice were treated by intratracheal instillation of 100 µg of TiO 2 NPs, CeO 2

Figure 2 :

 2 Figure 2: Morphometric analysis of the lung of the offspring of NPs-treated miceHistological examination was performed on hematoxylin and eosin stained sections of

Figure 3 :

 3 Figure 3: Silver element detection and localization in placenta and fetal lungsHematoxylin and eosin (H&E) staining and elemental XRF maps of sections of

Figure 4 :

 4 Figure 4: Expression of VEGF-α and MMP-9 in pup lungsVEGF-α mRNA expression (A) was significantly decreased in the TiO 2 NPs and CeO 2

Nanoparticles characteristics

  Nanoparticles characteristics: shape and size were determined by transmission electron microscopy (TEM), crystallinity of TiO 2 NPs and CeO 2 NPs was determined by X-ray diffractometer, specific surface area was estimated by TEM or using Brunauer Emmett Teller analysis, zeta potential and hydrodynamic diameter was determined by dynamic light scattering, NPs endotoxin content was examined using the Limulus Amebocyte Lysate test. TiO 2 : Titanium dioxide, CeO 2 : Cerium dioxide, Ag: Silver, PVP: polyvinylpyrrolidone.

	Composition Coating	Crystalline surface	Shape	Dimensions (nm)	Specific surface area (m 2 /g)	Zeta potential pH 7 (mV)	Hydrodynamic diameter (nm)	Endotoxin level
	TiO 2	No	Anatase	Spherical	12.3 ± 0.1	96 ± 2.3	3.7 ± 0.2	1280	0
	CeO 2	No	Cerianite	Spherical	22.4 ± 0.2	42 ± 0.5	9.5 ± 0.6	1480	0
	Ag	PVP	Amorphous Spherical	10.4 ± 0.1	60 ± 0.6	-6.2 ± 0.1	38 and 720	0

Table 2 : Lung volume and lung morphometry in the offspring at post-delivery day 14.5 and 49.5

 2

	Post-delivery day	14.5

n=10/group. Results are expressed as the median and interquartile range. Statistically significant differences between exposed and control groups are indicated as *: p<0.05, **: p <0.01, ***: p <0.001. Linear regression model adjusted for sex. TiO 2 : Titanium dioxide, CeO 2 : Cerium dioxide, Ag: Silver.

Table 3 : Element detection in the placenta and fetal lung at GD 17.5 using ICP-MS

 3 Placenta and fetal lung were obtained at gestational day (GD) 17.5, Median values (interquartile range) are reported. Statistically significant differences between exposed and control groups are indicated as **: p <0.01, ***: p <0.001. ICP-MS: Inductively Coupled Plasma Mass Spectrometry, Ti: Titanium, TiO 2 : Titanium dioxide, Ce: Cerium, CeO 2 : Cerium dioxide, Ag: Silver.

					Element concentration (µg/g)
			Exposure	Ti	Ce	Ag
	Placenta	Saline TiO 2 CeO 2 Ag	0.611 (0.256-1.431) 5.092 (1.624-5.513) ***	0.001 (0.001-0.001) 0.004 (0.001-0.007) **	0.001 (0.001-0.003) 5.484 (2.872-7.136) ***
			Saline	2.298 (2.013-3.082)	0.001 (0.001-0.001)	0.005 (0.001-0.026)
	Fetal	lung	TiO 2 CeO 2 Ag	1.477 (0.127-3.259)	0.001 (0.001-0.115)	0.054 (0.036-0.066) ***
	n ≥4/group,

Acknowledgments

We thank Dr. V. Cohignac for her help in the animal experiments. The expert technical assistance of M. Surenaud is also gratefully acknowledged. Special thanks to Dr. P. Andujar, Dr. P. Launois, S. Rouziere, Dr. T. Fournier, Dr. PH. Jarreau, S. Valentino, Dr. P.

Chavatte Palmer, Dr. A. Tarrade and Dr. J. Tran Van Nhieu for their helpful comments.

Funding

This work was carried out in the framework of the Labex SERENADE (ANR-11-LABX-0064) and A*MIDEX project (ANR-11-IDEX-0001-02), funded by the "Investissements d'Avenir" French Government program managed by the French National Research Agency (ANR). DHU ATV-B.

Declaration of interests

The authors declare that they have no competing interests.

Ethics approval and consent to participate

Ethical approval for all animal experimentation was obtained from the local ethics committee (N° 12-104, ComEth Afssa/ENVA/UPEC, Maisons-Alfort, France)