Chloé Cartier

Charline Warembourg

Christine Monfort

Florence Rouget

Gwendolina Limon

Gaël Durand

Sylvaine Cordier

Dave Saint-Amour
email: saint-amour.dave@uqam.cahighlights

Cécile Chevrier

Children's Contrast Sensitivity Function in Relation to Organophosphate Insecticide Prenatal Exposure in the Mother-Child PELAGIE Cohort

Keywords: Contrast Sensitivity, Vision, Organophosphate Insecticides, Development, Children

 Very few studies in children have investigated the potential impact of prenatal exposure to organophosphate pesticides (OP) on sensory functions  The association between urinary OP metabolite levels collected in pregnant women and child's visual contrast sensitivity was evaluated in 180 school-aged children from the mother-child PELAGIE cohort (France)  Although no associations were observed in the entire sample, maternal OP urinary metabolite levels were associated with a decrease in contrast sensitivity in boys only

Introduction

Organophosphate pesticides (OPs) are among the most widely used insecticides worldwide. These chemicals are used for conventional agriculture crops, residential pest control and veterinary purposes. In France, these chemicals have been found in dietary products [START_REF] Nougadere | Total diet study on pesticide residues in France: levels in food as consumed and chronic dietary risk to consumers[END_REF], outdoor air (Air Breizh 2012), and indoor environments.

Human biomonitoring studies have demonstrated widespread contamination [START_REF] Bradman | Measurement of pesticides and other toxicants in amniotic fluid as a potential biomarker of prenatal exposure: a validation study[END_REF][START_REF] Bradman | Variability of organophosphorous pesticide metabolite levels in spot and 24-hr urine samples collected from young children during 1 week[END_REF]. Indeed OP residues have been found on the hands of children and adults (Bouvier et al., 2006a[START_REF] Bouvier | Pesticide exposure of nonoccupationally exposed subjects compared to some occupational exposure: a French pilot study[END_REF], in urine from different populations, and in amniotic fluid and meconium [START_REF] Bradman | Measurement of pesticides and other toxicants in amniotic fluid as a potential biomarker of prenatal exposure: a validation study[END_REF][START_REF] Whyatt | Measurement of organophosphate metabolites in postpartum meconium as a potential biomarker of prenatal exposure: a validation study[END_REF][START_REF] Whyatt | Biomarkers in assessing residential insecticide exposures during pregnancy and effects on fetal growth[END_REF]. Population exposure to OP has been suggested to occur mainly via dietary intake of non-organic contaminated products [START_REF] Abb | Analysis of pesticides and PCBs in waste wood and house dust[END_REF][START_REF] Lu | Dietary intake and its contribution to longitudinal organophosphorus pesticide exposure in urban/suburban children[END_REF], but domestic use of insecticides and proximity to agricultural spraying areas were also found to be potential exposure sources [START_REF] Whyatt | Biomarkers in assessing residential insecticide exposures during pregnancy and effects on fetal growth[END_REF]. Breastfeeding and repeated hand-to-mouth behaviours are two other pathways for OP exposure in infants and children [START_REF] Bedi | Pesticide residues in human breast milk: risk assessment for infants from Punjab, India[END_REF][START_REF] Weldon | A pilot study of pesticides and PCBs in the breast milk of women residing in urban and agricultural communities of California[END_REF].

OPs were synthesized to eliminate insects through acetylcholine toxicity [START_REF] Costa | Current issues in organophosphate toxicology[END_REF].

OPs inhibit acetyl cholinesterase action that results in an increase in acetylcholine levels at synaptic junctions. OPs can also disrupt serotoninergic and dopaminergic pathways, as well as neuronal replication, differentiation and apoptosis during developmental processes [START_REF] Costa | Current issues in organophosphate toxicology[END_REF][START_REF] Slotkin | Organophosphate insecticides target the serotonergic system in developing rat brain regions: disparate effects of diazinon and parathion at doses spanning the threshold for cholinesterase inhibition[END_REF]. OP acute poisoning consequences are well identified but the impact of chronic low dose exposure, as observed in the general population, is still under investigation. Because the brain is more vulnerable to neurotoxic insult during development than later on [START_REF] Grandjean | Developmental neurotoxicity of industrial chemicals[END_REF][START_REF] Rice | Critical periods of vulnerability for the developing nervous system: evidence from humans and animal models[END_REF], the fetus, newborns and children are potentially the most vulnerable to OP developmental neurotoxicity while prenatal exposure may constitute a window of highest susceptibility.

Prenatal OP exposure has been associated with neurodevelopmental outcomes in several longitudinal birth cohort studies [START_REF] Gonzalez-Alzaga | A systematic review of neurodevelopmental effects of prenatal and postnatal organophosphate pesticide exposure[END_REF]. Adverse associations in children were observed between prenatal exposure to OP and fine motor skills [START_REF] Handal | Occupational exposure to pesticides during pregnancy and neurobehavioral development of infants and toddlers[END_REF], mental and psychomotor development [START_REF] Engel | Prenatal exposure to organophosphates, paraoxonase 1, and cognitive development in childhood[END_REF][START_REF] Eskenazi | Organophosphate pesticide exposure and neurodevelopment in young Mexican-American children[END_REF][START_REF] Llop | Prenatal and postnatal insecticide use and infant neuropsychological development in a multicenter birth cohort study[END_REF][START_REF] Rauh | Impact of prenatal chlorpyrifos exposure on neurodevelopment in the first 3 years of life among inner-city children[END_REF], visuospatial performance (Grandjean et al., 2006;[START_REF] Harari | Neurobehavioral deficits and increased blood pressure in school-age children prenatally exposed to pesticides[END_REF], intellectual functions [START_REF] Bouchard | Prenatal exposure to organophosphate pesticides and IQ in 7-year-old children[END_REF][START_REF] Engel | Prenatal exposure to organophosphates, paraoxonase 1, and cognitive development in childhood[END_REF][START_REF] Rauh | Seven-year neurodevelopmental scores and prenatal exposure to chlorpyrifos, a common agricultural pesticide[END_REF], but see Cartier et al., 2016;Donauer et al., 2016 for contrasting results), as well as symptoms of pervasive developmental disorder [START_REF] Eskenazi | PON1 and neurodevelopment in children from the CHAMACOS study exposed to organophosphate pesticides in utero[END_REF] and attention deficit/hyperactivity disorder [START_REF] Marks | Organophosphate pesticide exposure and attention in young Mexican-American children: the CHAMACOS study[END_REF].

Very few studies have investigated the potential impact of prenatal exposure to OP on the integrity of sensory function. This issue needs to be addressed because sensory function is crucial for intellectual or cognitive development, and sensory assessments can potentially reveal brain functional abnormalities that are not detectable with standard cognitive evaluations. The traditional method to assess visual function is acuity testing, which

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measures the ability to recognize smaller and smaller stimuli at maximal contrast (e.g., a black letter or any other shape on a white background). Because visual acuity is easily disrupted by refraction errors in the eye and it relies only on high contrast and spatial resolution stimuli, this method may be insensitive to reveal deficits of retinal and/or postretinal processing, such as the vision loss at low spatial resolution caused by neural defects.

Moreover, vision in everyday life is rarely under black-and-white (maximal) contrast. This is why an alternative way to assess vision is through the ability to see a shape or pattern of decreasing contrast (light intensity) against a background. Thus the visual discrimination is tested around the threshold level where the signal (e.g., pattern) is embedded in internal noise (e.g., greater spontaneous neural activity), so that contrast sensitivity represents a discrimination measure of the signal relative to the noise in the visual system [START_REF] Silvestre | Internal noise sources limiting contrast sensitivity[END_REF]. In fact, assessing visual function in the context of signal-to-noise ratio is the key feature of contrast sensitivity testing.

Because contrast sensitivity testing is typically repeated for several stimulus sizes (from large to small), it provides, in comparison to acuity, a more comprehensive and sensitive measure of visual function. This approach has been successfully used to reveal visual neurotoxicity in relation to exposure to several environmental chemicals (e.g., [START_REF] Burbacher | Prenatal methylmercury exposure affects spatial vision in adult monkeys[END_REF][START_REF] Cartier | Prenatal and 5-year p,p'-DDE exposures are associated with altered sensory processing in school-aged children in Nunavik: a visual evoked potential study[END_REF][START_REF] Costa | Long-term occupational exposure to organic solvents affects color vision, contrast sensitivity and visual fields[END_REF][START_REF] Fillion | Toxic risks and nutritional benefits of traditional diet on near visual contrast sensitivity and color vision in the Brazilian Amazon[END_REF][START_REF] Frenette | Contrast-sensitivity loss in a group of former microelectronics workers with normal visual acuity[END_REF][START_REF] Indhushree | Visual functions of workers exposed to organic solvents in petrochemical industries[END_REF][START_REF] Jimenez Barbosa | Exposure to organic solvents used in dry cleaning reduces low and high level visual function[END_REF][START_REF] Lebel | Evidence of early nervous system dysfunction in Amazonian populations exposed to low-levels of methylmercury[END_REF][START_REF] Oliveira | Organic solvent exposure and contrast sensitivity: comparing men and women[END_REF][START_REF] Reif | Neurobehavioral effects of exposure to trichloroethylene through a municipal water supply[END_REF][START_REF] Rice | Comparison of visual function at adulthood and during aging in monkeys exposed to lead or methylmercury[END_REF][START_REF] Saint-Amour | Alterations of visual evoked potentials in preschool Inuit children exposed to methylmercury and polychlorinated biphenyls from a marine diet[END_REF][START_REF] Schreiber | Apartment residents' and day care workers' exposures to tetrachloroethylene and deficits in visual contrast sensitivity[END_REF][START_REF] Till | Vision abnormalities in young children exposed prenatally to organic solvents[END_REF]. Interestingly, [START_REF] Frenette | Contrast-sensitivity loss in a group of former microelectronics workers with normal visual acuity[END_REF] have shown that microelectronic workers exposed to solvents have lower contrast sensitivity in comparison to the control group, but normal visual acuity.

To our knowledge, only a single study in children has reported visual function alterations in relation to OP prenatal exposure. [START_REF] Handal | Occupational exposure to pesticides during pregnancy and neurobehavioral development of infants and toddlers[END_REF] showed that prenatal exposure to OP from maternal occupation in the cut-flower industry during pregnancy was associated with lower visual acuity in Ecuadorian infants and toddlers. However, the OP concentrations were not directly measured in this study, so that the decrease in child acuity might not be associated with prenatal exposure to OP per se, but to other flower-industry-

Material and methods

Participants

The PELAGIE cohort was initiated to evaluate the potential impact of prenatal exposure to environmental chemicals on pregnancy outcomes and child development. Between May 2002 andFebruary 2006, 3421 pregnant women from Brittany were recruited before 19 weeks of amenorrhea [START_REF] Chevrier | Urinary biomarkers of prenatal atrazine exposure and adverse birth outcomes in the PELAGIE birth cohort[END_REF][START_REF] Petit | Association of environmental insecticide exposure and fetal growth with a Bayesian model including multiple exposure sources: the PELAGIE mother-child cohort[END_REF][START_REF] Petit | Impact on fetal growth of prenatal exposure to pesticides due to agricultural activities: a prospective cohort study in Brittany, France[END_REF].

Questionnaires at inclusion were administered to document social, demographic, occupational, and medical family characteristics, dietary habits (fish, fruit and vegetable consumption), and lifestyle. For urinary assays a subcohort of 601 women (18% of the total cohort) was randomly selected from the cohort members who gave birth to a live-born singleton [START_REF] Chevrier | Urinary biomarkers of prenatal atrazine exposure and adverse birth outcomes in the PELAGIE birth cohort[END_REF].

Children at 6 years of age were invited to participate in a neuropsychological follow-up study, that included an evaluation of their contrast sensitivity visual function and cognitive function. Exclusion criteria were the following: birth before 35 weeks of gestation, adverse findings at delivery (e.g., hypoglycemia, 5-minute Apgar score <7), neonatal hospitalization or resuscitation, genetic anomalies, and maternal or child death. Maternal urinary samples and children's vision evaluations were available for 185 of the 243 children who participated in this 6-year-old follow-up study (visual testing material was available for 76.5% of the home visits). Among them 5 children were excluded: 4 children did not wear their glasses at the testing time and one child had nystagmus. All subjects gave informed consent before participating in this study. The appropriate French ethics committees approved all study procedures, including the informed consent forms.

Measurements of OP exposure

Functional Acuity Contrast Test

The Functional Acuity Contrast Test (FACT®) is a chart with five rows of vertical gratings embedded in circular patches (1.7 degrees of visual angle), each having a specific spatial frequency, that is 1.5 cycles per degree (cpd) in row A, 3 cpd in row B, 6 cpd in row C, 12 cpd in row D, and 18 cpd in row E. In each row, the grating contrast uniformly decreases in nine levels by 0.15 log units from high (left side) to low (right side) contrast levels (Fig. 1). The FACT was used to assess the children's visual function. Assessments were conducted at home in a normal room illumination by one of two trained psychologists, blind to the child's prenatal and current OP exposure. Ambient light (illumination) was measured just before the test. The child was asked to indicate the grating orientation (left, up or right) for each stimulus. The test was presented monocularly at a distance of 45 cm to the best eye according to the child's preference. The child was instructed to use glasses during the test if normally worn. The child scores, one per row or spatial frequency, were the last correct answer given, so that higher scores indicate better performance. The FACT was administrated twice; a practice session followed by the actual test.

Potential covariates

A maternal interview was also conducted at home during the 6-year-old follow-up visit, with subtests of the Wechsler Adult Intelligence Scale (WAIS-III) to document the mothers' verbal intellectual quotient (IQ). The Home Observation for Measurement of the Environment questionnaire (HOME) [START_REF] Caldwell | Home observation for measurement of the environment[END_REF] was also used to assess the family environment.

Several variables were documented with questionnaires at enrollment: Mother's age, education level (university level or lower), tobacco use (yes or no) and alcohol use (yes or no) during pregnancy, fish consumption during pregnancy (≥ 2 per week or less), gestational age, parity (0 or ≥ 1). Additional variables were documented at the 6-years neuropsychological follow up: Mother verbal IQ, HOME score, breastfeeding (yes or no), duration of video gaming (0, 0-1.5, ≥ 1.5 h per week), duration of television watching (<2.5, 2.5-4.5, >4.5 h per week), extra-curricular sports activities (no, yes), sleep duration (10.5, 10.5-11, >11 h per day), the child's examiner (psychologist 1 or 2), and ambient light intensity (in lux units) at the time of testing.

Statistical analysis

We used linear regression models to assess the relation between maternal urinary concentrations of OP metabolites and children's FACT scores (row A-lowest spatial frequency to row E-highest spatial frequency). The DAP, DM, and DE metabolites were considered in three separate independent models. All variables associated with both prenatal OP metabolite urinary concentrations and FACT scores at p ≤ 0.2 were considered as potential confounding factors and included in the final regression model (i.e., maternal education level, child's sex, breastfeeding, and duration of television watching).

The variables highly associated (p ≤ 0.05) with FACT scores only were also included in the final regression models (i.e. total HOME score, ambient light, extra-curricular sports activities, and child's interviewer). To account for the potential role of current OP exposure on FACT scores, the children's OP metabolite urinary concentrations were entered in final

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regression models. Analyses were conducted for boys and girls separately, but also for the whole sample. Although testing ambient light was optimal for most of children (see Table 1), the range was from 20 to 7880 lux due to various conditions in which the test was administrated at home (room with or without windows, etc.). Because low lighting testing may reduce visual performance, a sensitivity analysis was conducted by re-running the regression models without participants for which the FACT was conducted in non-optimal lighting conditions. All analyses were performed with SPSS software (version 20).

Results

Descriptive characteristics of the participants are presented in Table 1. Girls and boys were equally represented in the sample (48.9% and 51.1%, respectively). Mothers' age at the time of study inclusion ranged from 22 to 44 years and most (65.6%) were highly educated (university level). Occasional alcohol drinking (at least once a week) was reported by 12.8% of the pregnant women, and 23.3% reported smoking at the beginning of pregnancy.

The average gestation age was 39.5 weeks (median = 40 weeks) with a range of 36 to 41 weeks. Finally, a majority of children (65%) were breastfed, but less than 6 weeks for most of them.

Urinary concentrations of DAP metabolites are presented in Table 2. DAP, DM, and DE metabolites were quantified in 91.7%, 90%, and 49.4% of the prenatal maternal urinary samples, respectively. Among quantified values median urinary concentrations were 47.9 nmol/L for DAP, 39.45 nmol/L for DM, and 13.3 nmol/L for DE. DAP, DM, and DE metabolites were detected in 81.1%, 63.9% and 53.9% of the children's urinary samples, respectively. Median urinary concentrations among detected values were 21 nmol/L for DAP, 15.2 nmol/L for DM, and 10.8 nmol/L for DE. These values are lower than DAP urinary concentrations measured in other North America longitudinal birth cohorts (Cartier et al., 2016). Descriptive characteristics of FACT scores are presented in Table 3, for all children and for girls and boys separately. Scores did not differ between sex, except for row E where boys had statistically significant better scores than girls (2.84 vs. 2.16, p value = 0.04). nmol/L)=-0.44, 95% CI: -1.45; 0.57) (Figure 2). Among girls, almost all estimates were positive and displayed no monotonic dose-response relationship. Statistically significant estimates were observed between intermediate prenatal urinary concentrations of DAP and FACT scores at 3 cpd (β=0.88, 95% CI: 0.02; 1.74) and 6 cpd (β=0.88, 95% CI: 0.03; 1.72), as well as between intermediate prenatal urinary concentrations of DM and FACT scores at 6 cpd (β=0.93, 95% CI: 0.16; 1.69) and 12 cpd (β=1.11, 95% CI: 0.18; 2.04) (Figure 2).

No statistically significant association was observed between prenatal total DAP, DE or DM urinary levels and FACT scores for all children as a whole (see Table 4, supplement material).

When considering child OP exposure, no significant association was found between OP urinary metabolite concentrations (DAP, DE, DM) measured at 6 years of age and FACT scores for boys (Figure 3). Significant associations were found for girls between DM urinary concentrations and better FACT scores for the mid-range spatial frequency 6 cpd (<5.24 nmol/L=ref; β(5.24-29.75 nmol/L)=0.66, 95% CI: -0.07; 1.38; β(>29.75 nmol/L)=0.86, 95% CI: 0.85; 1.64) and for the highest spatial frequency 18 cpd (<5.24 nmol/L=ref; β(5.24-29.75 nmol/L)=1.09, 95% CI: 0.12; 2.05; β(>29.75nmol/L)=1.08, 95% CI: 0.05; 2.11) (Figure 3). No association was found for the whole sample (see Table 5, supplement material).

A sensitivity analysis was conducted by re-running the regression models without participants for which the FACT was conducted in low light conditions. Children who was

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not tested with at least 55 lux (n=24), which is about the minimal illumination level for photopic or daylight vision, were thus excluded for this analysis. Results revealed no change in the associations reported above (data not shown).

Discussion

To our knowledge the present study is one of the first to assess the relation between OP prenatal exposure and visual function in school-aged children using urinary biomarkers of exposure and standardized visual tests in the general population. After controlling for confounding factors no association was observed between maternal DAP, DM or DE urinary levels and children's FACT scores. In boys, prenatal DE levels were significantly associated with a decrease in contrast sensitivity scores in moderate-to-high spatial frequencies (6 and 12 cpd), while better contrast sensitivity scores to the same spatial frequencies were observed among girls in association with prenatal DM urinary concentrations with a non-monotonous relation.

Alterations of visual functions in relation to exposure to other toxic agents have been reported in several human studies, in particular for solvents. In a population of 182 adult workers exposed to organic solvents, Gong et al. reported significantly lower contrast sensitivity than those in the control group but only at spatial frequencies of 6 and 12 cpd; no significant differences were found at 1.5, 3 or 18 cpd. The sex of the subjects was not detailed in this article. In regards to prenatal exposure, gestational exposure to tetrachloroethylene from drinking water has also been associated with lower FACT scores at intermediate and high spatial frequencies in adults, although a statistically significant difference was found only at 18 cpd [START_REF] Getz | Prenatal and early childhood exposure to tetrachloroethylene and adult vision[END_REF]. In children, [START_REF] Till | Assessment of visual functions following prenatal exposure to organic solvents[END_REF] showed, through electrophysiological measures, abnormal chromatic responses and reduced contrast sensitivity in a 2.5-year-old boy following prenatal exposure to tetrachloroethylene. In a subsequent study, on 21 infants born to women who were occupationally exposed to solvents during pregnancy [START_REF] Till | Vision abnormalities in young children exposed prenatally to organic solvents[END_REF] Our findings are in line with previous studies investigating the association between prenatal exposure to OP and visual function. In a cross-sectional study in Ecuador, prenatal occupational exposure to OP pesticides during pregnancy was evaluated through maternal self-reported questionnaires and linked to the visual acuity of the infants and young children [START_REF] Handal | Occupational exposure to pesticides during pregnancy and neurobehavioral development of infants and toddlers[END_REF]. Visual acuity deficits were found in children from 9 months to 8 years of age whose mothers were occupationally exposed to pesticides during their pregnancy, compared to children whose mothers were not. The authors reported that in these local greenhouses, OP insecticides are the most widely used pesticides, suggesting that prenatal exposure to OP might play an adverse role in children's visual function integrity, as reported in the present study. Of note, recent data collected from a small number of 9-month-old infants in China (n = 27) suggest slower auditory brainstem processing in relation to prenatal exposure to pesticides [START_REF] Sturza | Prenatal exposure to multiple pesticides is associated with auditory brainstem response at 9months in a cohort study of Chinese infants[END_REF], suggesting that the toxicity of OP on sensory function may not be restricted to the visual modality.

It is generally thought that deficits in contrast sensitivity observed at low-to-medium spatial frequencies mainly reflect defects in post-retinal neural processing, whereas those at high frequencies (and therefore fine spatial vision) are more likely due to the quality of the optics of the eye [START_REF] Waksman | Contrast sensitivity in occupational and environmental neurotoxicology: what does it really mean?[END_REF]. The OP-related reduced scores of contrast sensitivity observed in boys occurred for medium (6-12 cpd) spatial frequency gratings, suggesting alterations of neural processing along the visual pathway. Unexpectedly, better performances of contrast sensitivity in association with intermediate levels of prenatal DM urinary concentrations were observed in girls. We have no explanation for these results, although we cannot exclude the possibility of remaining confounding factors or chance findings. However, sex-dependent effects have been previously observed in studies investigating child neurodevelopment in relation to OP exposure. In a rural California longitudinal birth cohort, prenatal OP exposure was associated with child behavioural problems with a stronger association among boys [START_REF] Marks | Organophosphate pesticide exposure and attention in young Mexican-American children: the CHAMACOS study[END_REF]. Similarly, memory deficit was observed only in boys in association with prenatal exposure to OP in another North American longitudinal cohort from New York, with no apparent deficit in girls [START_REF] Horton | Does the home environment and the sex of the child modify the adverse effects of prenatal exposure to chlorpyrifos on child working memory?[END_REF]. Because genetic and hormonal systems differ between boys and girls, Our results suggest the importance of assessing the functional integrity of sensory processing when evaluating chemical neurodevelopmental toxicity. Since visual processing precedes cognitive and behavioural response processing, it is possible that neuropsychological performance decrements observed by previous studies in children exposed to OPs are not fully explained by alterations of cognitive processes occurring in high-level brain areas, but may also result, at least in part, from OP sensory alterations.

Visual assessment in epidemiological cohort studies, in addition to neuropsychological testing, allow adjustments for potential visual deficits and, therefore, a better prediction of cognitive dysfunction in association with exposure, as shown in the Faroese children in relation to mercury exposure [START_REF] Grandjean | Impact of contrast sensitivity performance on visually presented neurobehavioral tests in mercury-exposed children[END_REF]. Moreover, evaluating sensory processes might also reveal subtle functional changes in the brain that are not necessarily detectable with cognitive evaluations.

One major limitation of our study concerns OP exposure assessment. OPs are characterized by a short biological half-life and are rapidly metabolized and excreted in urine [START_REF] Needham | Assessing exposure to organophosphorus pesticides by biomonitoring in epidemiologic studies of birth outcomes[END_REF]. A single urine sample as used here is more likely to represent recent OP exposure than chronic cumulative exposure; repeated urinary measures might have enabled better characterization of OP exposure, especially among children [START_REF] Bradman | Variability of organophosphorous pesticide metabolite levels in spot and 24-hr urine samples collected from young children during 1 week[END_REF][START_REF] Griffith | Organophosphate pesticide metabolite levels in pre-school children in an agricultural community: within-and between-child variability in a longitudinal study[END_REF]. Moreover, the existence of preformed DAP residues found in food reduces the specificity of our exposure assessment, which makes it impossible to distinguish between exposure to the active parent compound from exposure to the inactive metabolite itself in urinary DAP concentrations [START_REF] Lu | Dietary intake and its contribution to longitudinal organophosphorus pesticide exposure in urban/suburban children[END_REF][START_REF] Zhang | Dialkylphosphates (DAPs) in fruits and vegetables may confound biomonitoring in organophosphorus insecticide exposure and risk assessment[END_REF]. Indeed measurement of DAP residues in fruits and vegetables showed more preformed OP metabolites than parent compounds in the majority of the samples tested [START_REF] Zhang | Dialkylphosphates (DAPs) in fruits and vegetables may confound biomonitoring in organophosphorus insecticide exposure and risk assessment[END_REF]. Despite these limitations, a urinary biomarker is a simple, noninvasive tool for assessing prenatal OP exposure, and has been shown, in previous studies, to be effective in detecting OP developmental neurotoxicity [START_REF] Bouchard | Prenatal exposure to organophosphate pesticides and IQ in 7-year-old children[END_REF][START_REF] Engel | Prenatal exposure to organophosphates, paraoxonase 1, and cognitive development in childhood[END_REF][START_REF] Rauh | Seven-year neurodevelopmental scores and prenatal exposure to chlorpyrifos, a common agricultural pesticide[END_REF]. Our results are also limited by sample size, in considering statistical analysis administration took place at the children's homes, natural variability in the illumination conditions was observed. This variation in illumination was controlled for in our regression models. Nonetheless in order to reduce measurement errors in future studies and to have a better estimate of the influence of OP on CS function it would be helpful to conduct assessments in the same illumination condition for all children using an ophthalmic light for instance.

Conclusion

In conclusion, our study showed associations between prenatal biomarkers of OP exposure and children's FACT scores, pointing out the relevance of using standardized visual assessments in cohort studies to better characterized the impact environmental contaminant on child's visual integrity. Replication of these findings is required and further epidemiological and laboratory studies are necessary to make reasonable causal inferences between OP exposure and visual function.

  related hazards. The present study aims to examine associations between prenatal OP exposure, measured from OP urinary metabolite samples, and school-age children's visual function, in the French mother-child cohort PELAGIE.

  At enrollment, i.e., before 19 weeks of amenorrhea, a first morning void maternal urine sample was collected at home into 10-mL vials containing nitric acid to prevent bacterial multiplication. Pregnant women returned the urine sample to the research laboratory by local mail in a self-addressed stamped package. At reception, samples were frozen at -20 °C until shipment to the analysis laboratory. Children at age 6 were asked to provide the first-morning void urine sample on the day of the home visit, which was retrieved by the psychologist at that time. First morning void urine samples were chosen as the most concentrated urine samples.Each urine sample, one from the mother and one from the child, was analyzed for six nonspecific dialkylphosphate (DAP) metabolites of numerous OP insecticides (diethylphosphate (DEP), diethylthiophosphate (DETP), diethyldithiophosphate (DEDTP), dimethylphosphate (DMP), dimethylthiophosphate (DMTP) and dimethyldithiophosphate (DMDTP)). The LABOCEA Institute performed the chemical analyses (Plouzané, France). The maternal samples were analyzed by liquid chromatography (Alliance Waters, Separations Module 2690), with a Synergi Fusion RP C18 column, (250 × 2 mm, 4 μm) and triple quadrupole mass spectrometry (LC/MSMS), followed by solid phase extraction (Symbiosis Prospekt II type, Hysphere C18 HD cartridge). Detection relied on LC/MSMS (Quattro Ultima, Micromass/Waters). Two internal standards were used for extraction and detection controls: deuterated diuron 6 and Di-n-butylphosphate, provided by Riedel-de-Haën Fine Chemicals and Dr. Ehrenstorfer GmBH. The children's 1-mL samples were analyzed by solid phase extraction and liquid chromatography (Waters Acquity UPLC), with a Waters BEH C18 column (150 × 2.1 mm, 1.7 μm) and triple quadrupole mass spectrometry (UPLC/MSMS) detection after online solid phase extraction (Waters 2777C and Waters Oasis HLB Direct Connect cartridges). UPLC/MSMS was used for detection (Xévo TQ-S, Waters). Reference standards were di-ethylthiophosphate D10 and dimethylthiophosphate D6 (provided by Sigma-Aldrich and Dr. Ehrenstorfer GmBH).Additional details were provided in the available supplemental data inCartier et al. (2016).The limits of quantification (LOQ) for the chemical analyses of maternal urine samples were 1.25, 1.7, 0.02, 0.2, 1, and 0.45 μg/L for DEP, DETP, DEDTP, DMP, DMTP, and A DMDTP, respectively. For the children's samples, values between the limit of detection (LOD) and the LOQ were available. The LODs were 0.2, 0.1, 0.005, 0.06, 0.32, and 0.13 μg/L for DEP, DETP, DEDTP, DMP, DMTP, and DMDTP, respectively. No imputations were made for censored values (i.e., <LOQ for maternal urine samples or <LOD for children's samples). Metabolite concentrations in micrograms per liter were converted to their molar concentrations (nanomoles per liter). DEP, DETP, and DEDTP concentrations were summed to obtain overall concentrations of diethylphosphate metabolites (DE), which are metabolites of the ethyl group of OP (Chlorpyrifos, Diazinon). DMP, DMTP, and DMDTP were summed to obtain dimethylphosphate metabolites (DM), which are metabolites of the methyl group of OP (Dichlorvos, Malathion). DAP were the sum of all six metabolites (sum of DE and DM). The urinary concentrations were then grouped into three exposure categories: 1. censored values, 2. uncensored values ≤ median, 3. uncensored values > median for DE (<66.7% of uncensored values), and in tertiles for DAP and DM (>66.7% of uncensored values).

  OP urinary metabolite levels assessed during pregnancy and children's 6-year FACT scores were evaluated using linear regression models for boys and girls separately. When considering boys only, prenatal urinary DE concentrations were significantly associated with a decrease in FACT scores with the highest category level at 6 cpd (<LOQ=ref; β(LOQ-13.3 nmol/L)=-0.63, 95% CI: -1.64; 0.38; β(>13.3 nmol/L)=-1.38, 95% CI: -2.48; -0.29) and 12 cpd (<LOQ=ref; β(LOQ-13.3 nmol/L)=0.06, 95% CI: -0.91; 1.03; β(>13.3 nmol/L)=-1.44, 95% CI: -2.48; -0.39). Moreover, a significant negative association between prenatal urinary concentrations of DAP and FACT scores was found at 12 cpd (<27.7 nmol/L=ref; β(27.7-74.35 nmol/L)=-1.04, 95% CI: -2.05; -0.03; β(>74.35

  , similar results were observed. That is, in comparison to age-matched controls, significantly lower visual acuity, abnormal chromatic responses (red-green vision) and significantly reduced contrast sensitivity scores in the low and intermediate spatial frequency range (< 10 cpd).

  have played a role in such gender-related differences in OP developmental neurotoxicity.

  sex; and we also cannot exclude the possibility of chance findings particularly for DE which had the highest frequency of non-detects. Because FACT

Figure 1 .

 1 Figure captions

Figure 2 .

 2 Figure 2. Associations between OP prenatal urinary metabolites and FACT scores.

Figure 3 .

 3 Figure 3. Associations between 6-years urinary metabolites and FACT scores.

Acknowledgments:

We are grateful to the gynecologists, obstetricians, ultrasonographers, midwives, pediatricians, psychologists, and families who participated in the study. We particularly thank Véronique Villalon, Catherine Nouyrigat and Jo Ann Cahn for their help and Mathilde Mordelet and Olivia Martin for the neuropsychological and visual testing. GL and GD are employed by the LABOCEA Company, Plouzane, France. This study received funding from the French National Research Agency (ANR-2010-PRSP-007) and the Fondation de France (FdF).

Conflict of interest statement:

The authors declare that they have no conflict of interest.