Prenatal and postnatal exposure to organophosphate pesticides and childhood neurodevelopment in Shandong, China

Environment International 108 (2017) 119–126

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Prenatal and postnatal exposure to organophosphate pesticides and childhood neurodevelopment in Shandong, China

MARK

Yiwen Wanga,b, Yan Zhangc, Lin Jic, Yi Huc, Jingjing Zhangc, Caifeng Wangc, Guodong Dingd, Limei Chene, Michihiro Kamijimaf, Jun Ueyamag, Yu Gaoc,⁎, Ying Tiana,c,⁎⁎ a MOE and Shanghai Key Laboratory of Children's Environmental Health, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China b Department of Neonatology, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China c Department of Environmental Health, School of Public Health, Shanghai Jiao Tong University School of Medicine, Shanghai, China d Department of Pediatrics, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China e Basic Medicine Faculty, Wuxi Medical School, Jiangnan University, Wuxi, Jiangsu Provience, China f Department of Occupational and Environmental Health, Nagoya City University Graduate School of Medical Sciences, Nagoya, Aichi 467-8601, Japan g Department of Pathophysiological Laboratory Sciences, Field of Radiological and Medical Laboratory Sciences, Nagoya University, Nagoya, Aichi 461-8673, Japan

A R T I C L E I N F O

A B S T R A C T

Keywords: Organophosphate pesticides Neurodevelopment Prenatal exposure Postnatal exposure China

Background: Although studies in laboratory animals demonstrate neurodevelopmental deficits caused by prenatal or postnatal organophosphate pesticide (OP) exposure, there is limited evidence on effects induced by not only prenatal but also postnatal exposure of children to OPs. Methods: We measured diethylphosphate (DE), dimethylphosphate (DM), and total dialkylphosphate (DAP) metabolites in maternal and child urine at 12 and 24 months of age and examined their relationship with developmental quotients (DQs) in 12-month-old infants and 24-month-old children in Shandong, China. Results: The median concentrations of total DAP metabolites (DAPs) in child urine [371.97 nmol/g creatinine (12-month-old infants), 538.64 nmol/g creatinine (24-month-old children)] were higher than those in maternal urine (352.67 nmol/g creatinine). Prenatal OP exposure was negatively associated with 24-month-old children's DQs, especially among boys. A 10-fold increase in prenatal DEs and DAPs was associated with a 2.59- and 2.49point decrease in social domain DQ scores in 24-month-old children (n = 262), respectively. However, positive association of postnatal exposure to OPs and 24-month-old children's DQs was observed (n = 237). Neither prenatal nor postnatal exposure to OPs was related to 12-month-old infants' DQs. Conclusions: These data suggested that prenatal OP exposure could adversely affect children's neurodevelopment at 24 months of age, especially among boys. The prenatal period might be a critical window of OP exposure. In view of the positive association with postnatal OP exposure, it is necessary to interpret findings with caution.

1. Introduction The exposure of general population to organophosphate pesticides (OPs) through environmental pollution and consumption of the pesticide-contaminated food and water has become a global issue. In China, > 300,000 tons of pesticides are used every year in agriculture, with OPs comprising approximately 70% of all pesticides used (Agriculture Information Network, 2006). Several studies have reported widespread exposure to OPs in susceptible populations (children and pregnant women), suggesting that OP exposure in children and pregnant women is a critical public health issue that deserves greater

⁎

concern (Eskenazi et al., 2004, 2007; Wang et al., 2012; Whyatt et al., 2004; Ye et al., 2008, 2009). There is evidence that OPs could cross the placenta and blood–brain barrier, and they have been detected in amniotic fluid and meconium (Bradman et al., 2003; Whyatt and Bar, 2001); this indicates that fetuses could be exposed to OPs. Moreover, children and fetuses are thought to be highly vulnerable to OP toxicity, because the brain is in rapid development, and concentrations of protective enzymes that deactivate OPs are lower than those in adults (Holland et al., 2006). Prenatal and early postnatal periods are critical for neurodevelopment. Evidence from studies in animals demonstrated that prenatal or

Corresponding author. Correspondence to: Y. Tian, MOE and Shanghai Key Laboratory of Children's Environmental Health, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, 1665 Kongjiang Road, 200092 Shanghai, China. E-mail addresses: [email protected] (Y. Gao), [email protected] (Y. Tian). ⁎⁎

http://dx.doi.org/10.1016/j.envint.2017.08.010 Received 20 April 2017; Received in revised form 18 July 2017; Accepted 16 August 2017 0160-4120/ © 2017 Elsevier Ltd. All rights reserved.

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and occupational pesticide exposures. Gender of the neonate, gestational age, and birth weight were collected from hospital delivery logs and medical records (Ding et al., 2013).

early postnatal OP administration could adversely affect offspring's neurodevelopment (Dam et al., 2000; Eskenazi et al., 1999), such as producing changes in cognitive, motor, and sensory functions as well as the nervous system developmental processes (Levin et al., 2001; Rice and Barone, 2000; Venerosi et al., 2009). However, limited epidemiological investigations in human populations assessed childhood neurodevelopment after prenatal or early postnatal OP exposure, with controversial results. Eskenazi et al. (2007) found that prenatal exposure to OPs was negatively associated with scores in the Mental Development Index (MDI), but postnatal exposure to OPs was positively associated with measurements in the MDI at 24 months of age. However, Rauh et al. (2006) found lower Psychomotor Development Index (PDI) and MDI scores in children at 36 months of age exposed prenatally to higher chlorpyrifos concentrations, but not in 12- or 24-month-old children. More recently, one study conducted in Jiangsu, China (known as an agricultural area with heavily used OPs) showed that prenatal OP exposure was related to neurodevelopmental delay in the adaptive domain, and postnatal OP exposure might be related to neurodevelopmental delay in the social and motor domains in 24-month-old children in the agricultural area (Liu et al., 2016). Concerns have been raised over the adverse effect of OPs, but evidence from China is still lacking about the potential effects on childhood neurodevelopment at 12 and 24 months of age after not only prenatal but also early postnatal OP exposure. Therefore, we recruited pregnant women and children from the Laizhou Wan Birth Cohort (LWBC) study in Shandong, China, and by using the Gesell Developmental Schedules (GDS) [which have been validated against a Chinese reference population (Song and Zhu, 1987) and are widely used for assessing child development in China and other countries (Cui et al., 2001; Zhu et al., 2005)], we tested the hypothesis of whether prenatal or postnatal exposure to OPs is associated with childhood neurodevelopment at 12 and 24 months of age.

2.3. Biologic sample collection Spot midstream urine specimens were obtained from the pregnant women during hospital admission for delivery, from 12-month-old infants, and from 24-month-old children during a follow-up visit, with their parents using clean glass bottles. If the infant or child could not provide the sample during the visit, the parents were asked to take a glass bottle home to collect a spot urine sample on the next day and send it to the hospital. The urine samples were stored at − 80 °C until analysis of six DAP metabolites in the Shanghai Clinical Research Center (SCRC). 2.4. OP metabolites in urine We measured six DAP metabolites in urine specimens by using gas chromatography-mass spectrometry (GC-MS) (Ueyama et al., 2010). Six analytes were measured in each specimen: three diethyl (DE) phosphate metabolites—diethyldithiophosphate (DEDTP), diethylthiophosphate (DETP), and diethylphosphate (DEP); and three dimethyl (DM) phosphate metabolites— dimethyldithiophosphate (DMDTP), dimethylthiophosphate (DMTP), and dimethylphosphate (DMP). Briefly, the procedures included liquid–liquid extraction (diethyl ether/acetonitrile), derivatization (pentafluorobenzylbromide [PFBBr]) and clean-up (multi-layer column) for GC-MS analysis, starting from urine specimens. (Detailed method was described in Supplementary materials.) Quality control (QC) samples were analyzed along with the collected samples to ensure the accuracy of analytical methods and results. QC samples were prepared by using pooled urine (from eight healthy adult volunteers) inserted blindly among the study samples. The recoveries of DAPs ranged from 27% to 139.2%. For the within-run precision, percent of relative standard deviation (%RSD) ranged from 0.5 to 6.3%. For the between-run precision, the %RSD was between 3.0 and 17.8% (Supplementary Table 1). The limit of detection (LOD) and limit of quantitation (LOQ) were calculated based on the signal-to-noise ratio of 3 and 10. The LOQs were 0.14–0.2 μg/L for DEP, 0.2 μg/L for DETP, 0.3 μg/L for DEDTP, 0.43–0.6 μg/L for DMP, and 0.5–1.0 μg/L for DMTP and DMDTP. The LODs were 0.06 μg/L for DEP and DETP, 0.09 μg/L for DEDTP, 0.3 μg/L for DMDTP and DMTP, and 0.18 μg/L for DMP. We assigned a value of the LOD divided by the square root of two to metabolite concentrations below the LOD (Hornung and Reed, 1990). Every metabolite was converted from the untransformed concentration (μg/L) to its corresponding molar concentration (nmol/ L) (DEP = concentration/0.154 μg/nmol; DETP = concentration/ 0.170 μg/nmol; DMP = concentration/0.126 μg/nmol; DMTP = concentration/0.142 μg/nmol) (Arcury et al., 2006). To avoid errors introduced by diluted or concentrated urine specimens, DAP concentrations were standardized to the creatinine levels in urine. Creatinine levels in urine were detected with an automated chemistry analyzer (7100 Hitachi, Japan).

2. Methods 2.1. Study population The present study was a longitudinal birth cohort study in the southern coastal area of Laizhou Wan (Bay) of the Bohai Sea, Shandong province, China (LWBC). Information on recruitment, eligibility, data collection, and questionnaires used in the LWBC has been published elsewhere (Ding et al., 2013). Eligibility criteria included residence in the area for ≥3 years; ≥18 years of age; a singleton pregnancy; and no report of assisted reproduction, illicit drug use, AIDS or HIV infection, gestational or preexisting diabetes, or pregnancy-associated or chronic hypertension (Ding et al., 2013). From March 2011 to December 2013, 436 women were recruited when admitted in labor at the only hospital in the area and underwent urinary dialkylphosphate (DAP) metabolite detection (group 1); 297 12-month-old infants (68.1%) underwent neurodevelopmental assessment (subgroup 1); and 262 24-month-old children (60.1%) underwent neurodevelopmental assessment (subgroup 2). For postnatal exposure assessment, 235 12-month-old infants underwent both urinary DAP metabolite detection and neurodevelopmental assessment (subgroup 3); 237 24-month-old children underwent both urinary DAP metabolite detection and neurodevelopmental assessment (subgroup 4) (Fig. 1). Written informed consent was obtained from all participants. The Medical Ethics Committee had approved the study protocol (Ding et al., 2013).

2.5. Neurodevelopment assessment We conducted the version of the GDS for 0- to 3-year-old children (Beijing Mental Development Cooperative Group, 1985) to the 12month-old infants and 24-month-old children in this cohort. Chinese was used for assessment. In each of the four domains (social, language, adaptive, and motor), a developmental quotient (DQ) was assigned to each child. The testing of the children was performed blinded to exposure status. The standardized mean ( ± SD) of the DQ was 100 ± 15. A child with a DQ lower than 85 was considered to have a

2.2. Data collection In the hospital, participating women were interviewed by specially trained nurses, using a questionnaire on social demographics, medical and obstetric history, lifestyle, and information about maternal pesticide exposures, including indoor insecticide usage during pregnancy 120

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Fig. 1. Flow chart of the inclusion criteria of the subjects.

the relatively low or high creatinine levels affected the reliability. We also considered other prenatal pollutants (lead, mercury, pyrethroid pesticides, and perfluoroalkyl and polyfluoroalkyl substances), and we tested collinearity between prenatal OP exposure and other pollutants, using the variance inflation factor (VIF) in the model. All VIFs were < 5 and in an acceptable range. In addition, because OP effects may differ by gender (Liu et al., 2016), the possible association between OP exposure and neurodevelopment stratified by child sex was evaluated. Statistical analyses were carried out using SPSS software (SPSS 16.0).

high probability of some organic impairment (Knobloch and Pasamanick, 1974). The cut-off point for differentiating normal development from developmental delay was a score of 84 (Hudon et al., 1998). A trained pediatrician who completed formal training with qualifications at Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine conducted the testing to maximize both the validity of the interpretation and the reliability of the assessment. 2.6. Data analysis

3. Results

Descriptive statistics were calculated for the sociodemographic characteristics of this study population, DAP concentrations, and DQ scores. Because of low detection rates (< 5%) for DEDTP and DMDTP, they were not included in the statistical analysis. The distributions of DMs (by summing the molar concentration of DMP and DMTP), DEs (by summing the molar concentration of DEP and DETP), and total DAP metabolites (DAPs) (by summing the molar concentration of DMP, DMTP, DEP, and DETP) (nmol/g creatinine) were skewed, and logtransformed values were used for regression analysis. We conducted separate linear regression models for each domain to assess the association among DMs, DEs, DAPs, and GDS performance. First, we selected confounders from the literature, listed as followed: family income, parental education [≤ 9 years (middle school), 10–12 years (high school), ≥13 years (greater than high school or college)], marital status, smoking during pregnancy, pre-pregnancy body mass index (BMI), maternal intelligence quotient (IQ), maternal age, parity, gestational age, birth weight, and child's sex (Eskenazi et al., 2007; Guodong et al., 2012; Rauh et al., 2006; Wang et al., 2016). For further analysis, we included those variables in the final regression models if they were all associated (p < 0.2) with DQs in a univariate model. We included the same covariates (child gender, household monthly income, paternal education, smoking during pregnancy, maternal education, maternal IQ, and maternal age) in all regression models. All regression analyses were run using log-transformed, creatinine-adjusted values. We ran GAMs by fitting splines to test the linearity assumption, and we did not find any obvious non-linear relationship (data not shown). In sensitivity analyses, we analyzed the relationships of OP exposure and neurodevelopment after excluding children considered to have developmental delays, to investigate whether developmentally delayed children affected the reliability of the results. We used log-transformed, non-creatinine-adjusted values to re-run models to investigate whether

3.1. Main demographic characteristics of study population Table 1 describes the demographic characteristics of the study population. Among 436 pregnant women, maternal mean age was 28.24 ± 4.14 years. Maternal mean IQ was 98.11 ± 12.30; 67.7% of the husbands completed education at the level of high school or higher, and 49.6% of the women completed education at the levels of high school or higher. Of the mothers, 60.1% lived in households with a monthly salary of less than RMB (¥) 3000 yuan ($483.60). [4000 yuan ($644.80) is the median household monthly income in Shandong Province.] All the pregnant women reported no occupational OP exposure; 7.1% of the pregnant women reported exposure to OPs through pesticide factories in the vicinity of the residence (data not shown); 35.8% of the women or her family members used household pesticide during her pregnancy (including mosquito repellent, cockroach killer, mothproofing agent, rodenticide, termite control agents, and other special pesticides); 87.2% of the pregnant women consumed fruits ≥ 4 times weekly during pregnancy, and 89.2% of the pregnant women consumed vegetables ≥4 times weekly during pregnancy. Although few smoked during pregnancy, 31.7% of the women lived with a smoker. Of the neonates, 48.6% were female. The mean ( ± SD) birth weight was 3410.83 ± 488.34 g. The mean ( ± SD) gestational age was 39.22 ± 2.18 weeks. Only 2.1% of the newborns (n = 9) weighed < 2500 g at birth, and 3.9% of the newborns (n = 17) were preterm (< 37 weeks). These children were included in the analyses. We found no substantial differences in the sociodemographic characteristics among subgroup 1 (n = 297), subgroup 2 (n = 262), subgroup 3 (n = 235), subgroup 4 (n = 237), and the initially recruited subjects (group 1, n = 436), denoting that the studied cohort generally 121

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child urinary DAPs, DMs, and DEs (data not shown).

Table 1 Demographic characteristics of the study population. Characteristic Maternal characteristic Maternal age (years) < 30 30–34 ≥ 35 Maternal IQ Maternal education (years) ≤ 9 (middle school) 10–12 (high school) ≥ 13(greater than high school or college) Paternal education(years) ≤ 9 (middle school) 10–12 (high school) ≥ 13(greater than high school or college) Household monthly salary (RMB) < 3000 ($483.60) 3000–5000 ($483.60—806.00) > 5000 ($806.00) Smoking during pregnancy No Lived with smoker Yes Household pesticide use during pregnancya No Yes Fruits consumed weekly during pregnancy < 4 time ≥ 4 times Vegetables consumed weekly during pregnancy < 4 time ≥ 4 times Infant characteristic Gender Male Female Gestational age (weeks) < 37 ≥ 37 Birth weight (g) < 2500 ≥ 2500

3.3. The distribution of GDS DQ scores of children at 12 and 24 months of age

n = 436 (%)

Supplementary Table 2 shows the distribution of GDS DQ scores of children at 12 months and 24 months of age. For the 12-month infants (subgroup 1, n = 297), the mean DQ scores in the motor, adaptive, language, and social domains were 106.1 (SD = 8.5), 100.2 (SD = 6.2), 96.6 (SD = 6.3), and 99.2 (SD = 6.7), respectively. The number (frequency) of developmental delays in the four domains were 0 (0%), 0 (0%), 10 (3.4%), and 2 (0.7%), respectively. For the 24month children (subgroup 2, n = 262), the mean DQ scores in the motor, adaptive, language, and social domains were 105.1 (SD = 7.4), 104.9 (SD = 7.7), 97.8 (SD = 7.9), and 106.9 (SD = 8.7), respectively. The number (frequency) of developmental delays in the four domains were 1 (0.4%), 2 (0.8%), 6 (2.3%), and 0 (0%), respectively. All the children were included in our further analyses.

298 (68.4) 106 (24.3) 32 (7.3) 98.11(SD = 12.30) 220 (50.4) 115 (26.4) 101 (23.2) 141 (32.3) 172 (39.5) 123 (28.2) 262 (60.1) 134 (30.7) 40 (9.2)

3.4. Associations between prenatal DAP concentrations and GDS DQ scores at 12 and 24 months of age

294 (67.4) 138 (31.7) 4 (0.9)

Prenatal DEs and DAPs were significantly negatively associated with social domain DQ scores in 24-month-old children (Table 3). A 10-fold increase in prenatal DEs and DAPs was associated with a 2.59-point (95% CI: −4.71 to −0.46) and 2.49-point (95% CI: − 4.85 to − 0.14) decrease in social domain DQ scores, respectively. After stratifying by child gender, the inverse relationship of prenatal OP exposure and DQ scores in the social domain in children at 24 months still existed in boys but not in girls (Supplementary Table 3). Among 24-month-old boys, a 10-fold increase in prenatal DEs was related to a 3.20-point (95% CI: − 6.31 to − 0.10) decrease in social domain DQ scores. After removing 24-month-old children considered to have developmental delays, the negative relationship of prenatal OP exposure and DQ scores in the social domain still existed (data not shown). However, we did not find any significant associations between DMs, DEs, or DAPs in pregnant women and DQ scores in 12-month-old infants.

280 (64.2) 156 (35.8) 56 (12.8) 380 (87.2) 47 (10.8) 389 (89.2)

224 (51.4) 212 (48.6) 17 (3.9) 419 (96.1) 9 (2.1) 427 (97.9)

a Information regarding the household pesticide (including mosquito repellent, cockroach killer, mothproofing agent, rodenticide, termite control agents, and other special pesticides) used by the mother herself or other family members during pregnancy.

3.5. Associations between postnatal DAP concentrations and GDS DQ scores at 12 and 24 months of age Postnatal DMs and DAPs were significantly positively associated with adaptive domain DQ scores in 24-month-old children (Table 4). A 10-fold increase in DMs and DAPs in the urine of children aged 24 months was related to a 3.00-point (95% CI: 0.97 to 5.04) and 2.99point (95% CI: 0.58 to 5.40) increase in DQ scores in the adaptive domain, respectively. However, we did not find any significant associations between DMs, DEs, or DAPs in infants aged 12 months and DQ scores in 12-month-old infants.

reflects the original one.

3.2. The distributions of maternal and child urinary DAP concentrations The DAP concentrations measured in maternal and children's urine at 12 and 24 months of age, both unadjusted and adjusted for creatinine, are summarized in Table 2. The detection rates for most of the DAP were higher than 80%. DMP was the highest in both maternal and children's urine and reached 21.42 μg/g (mothers), 21.30 μg/g (12month-old infants), and 38.68 μg/g (24-month-old children). DEP in maternal and children's urine reached 11.41 μg/g (mothers), 11.68 μg/ g (12-month-old infants), and 16.65 μg/g (24-month-old children). The concentrations for DMDTP and DEDTP were not further analyzed because of low detection rates (< 5%). DAP concentrations in children at 12 and 24 months of age were higher than those in mothers, and there was an increase in DAP concentrations from 12 to 24 months of age. Fig. 2 shows the comparison of median concentration of DAP in mothers and children at 12 and 24 months of age (μg/g creatinine). The median concentrations of DAPs in child urine [371.97 nmol/g creatinine (12-month-old infants), 538.64 nmol/g creatinine (24-month-old children)] were higher than those in maternal urine (352.67 nmol/g creatinine) (data not shown). Maternal urinary DAPs, DMs, and DEs were not correlated with

4. Discussion 4.1. The distributions of maternal and child urinary DAP concentrations This study analyzed DAP concentrations in pregnant women and children at 12 and 24 months of age in LWBC. In our study, the detection rates for most of the DAP concentrations were higher than 80%. We also found the DAP concentrations in both 12-month-old infants and 24-month-old children were higher than those in pregnant women. Young children are more susceptible to pesticide exposure due to their unique physiological characteristics and activity patterns. They drink more milk, water, or fruit juice and eat more food than adults in proportion to body weight. They crawl and play on the floor as well as put things into mouths; thus, they might have more chance to be exposed to 122

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Table 2 Detection rate, range, and percentiles of DAP concentrations in maternal and child urine at 12 and 24 months of age. Metabolites

Maternal DMP DMTP DEP DETP

n

Detection rate (%)

Not adjusted for creatinine (μg/L) Creatinine adjusted (μg/g) Range

25th

50th

75th

95th

Range

25th

50th

75th

95th

436 436 436 436

95.0 86.5 98.9 97.9

< LOD–224.46 < LOD–74.57 < LOD–292.19 < LOD–49.38

3.52 0.45 2.34 0.33

9.85 0.78 5.17 0.78

19.48 1.88 12.84 2.38

67.19 7.88 60.96 9.31

< LOD–480.36 < LOD–106.34 < LOD–996.04 < LOD–102.24

8.60 1.17 5.96 1.01

21.42 2.24 11.41 1.93

44.43 4.57 24.38 4.59

157.11 15.55 101.34 14.59

Infant (12 months) DMP 235 DMTP 235 DEP 235 DETP 235

94.9 86.4 99.1 89.8

< LOD–408.09 < LOD–65.43 < LOD–49.72 < LOD–21.77

2.24 0.42 1.06 0.21

4.34 0.89 2.40 0.55

9.90 2.17 4.68 1.29

53.84 14.43 15.07 5.73

< LOD–744.58 < LOD–381.75 < LOD–269.24 < LOD–116.59

11.69 2.54 6.66 1.51

21.30 4.74 11.68 2.76

47.05 10.42 21.02 5.41

176.67 38.26 56.56 18.48

Child (24 months) DMP 237 DMTP 237 DEP 237 DETP 237

93.2 74.3 95.8 95.8

< LOD–185.74 < LOD–32.80 < LOD–105.02 < LOD–77.06

3.54 < LOD 1.60 0.39

9.08 0.78 4.02 0.97

19.02 1.99 9.94 2.71

59.53 10.13 33.37 7.56

< LOD–2084.09 < LOD–123.18 < LOD–259.45 < LOD–159.99

16.55 2.03 8.15 2.08

38.68 3.64 16.65 3.95

68.30 7.52 28.74 8.01

202.73 21.57 85.51 20.52

DMP: dimethylphosphate; DMTP: dimethylthiophosphate; DEP: diethylphosphate; DETP: diethylthiophosphate; < LOD, below the limit of detection.

Generation R study and 0.29 μg/g in the CHAMACOS study. This suggests that more attention should be paid to exposures to pregnant women in China, given the relatively high exposure levels (Bradman et al., 2005; Ye et al., 2008). Regarding the exposure levels of OPs in children, we found that the young children (both 12 months and 24 months of age) in this study also had higher median concentrations (μg/g) of DMP (one of the major DAP metabolites) [21.30 (12-month-old infants), 38.68 (24-month-old children)] than did children of 2–17 years in a German study (10.70) and those of 6–7 years in an Italian study (13.83), children of 12 months (5.39) and 24 months (6.67) in the CHAMACOS study, and children of 23–25 months in Shanghai (9.85) (Aprea et al., 2000; Becker et al., 2006; Bradman et al., 2011; Guodong et al., 2012) (Supplementary Table 5). This finding raises concerns about the adverse effect induced by OP exposure in children.

pesticide residues (Eskenazi et al., 2007; Zartarian et al., 2000). We also observed an increase in DAP concentrations from 12 months to 24 months of age, which would be likely to continue because the 24month-old children might be more interactive with the environment than 12-month-old infants, leading to higher exposure to pesticide residues. Urinary DAP metabolite levels represent exposure to parent OP pesticides and their metabolites from use of pesticides at work, in the surrounding environment, at home (indoors or in the garden), or from dietary intake (Koch et al., 2002; Lu et al., 2008; Morgan et al., 2005). In the present study, 87.2% of the pregnant women consumed fruits ≥ 4 times weekly during pregnancy, 89.2% consumed vegetables ≥ 4 times weekly during pregnancy, 35.8% of the pregnant women or her family members used household pesticide during her pregnancy. All the pregnant women reported no occupational OP exposure, although a few pregnant women reported exposure to OPs through pesticide factories in the vicinity of the residence (7.1%). Thus, in our study, the main exposure route for OPs was assumed to be through diet and household pesticide use. As shown in Supplementary Table 4, the maternal median concentrations of major DAP metabolites (DMP and DEP) in our study were higher than those reported in the developed countries. For DMP, they were 21.42 μg/g in the present study versus 17.64 μg/g in the Generation R study and 1.92 μg/g in the CHAMACOS study; for DEP, they were 11.41 μg/g in the present study versus 2.96 μg/g in the

4.2. Associations between prenatal DAP concentrations and GDS DQ scores at 12 and 24 months of age In our study, prenatal DEs and DAPs were related to lower social domain DQ scores in 24-month-old children rather than in 12-monthold infants. Most studies found that the age of appearance of the adverse effect of prenatal exposure to OPs on childhood neurodevelopment was 24 months and older (Eskenazi et al., 2007; Marks et al., 2010; Rauh et al., 2006). Because cascading developmental procedures

Fig. 2. Comparison of median concentration of DAP in mothers and children at 12 and 24 months of age (μg/g creatinine).

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Table 3 Association between maternal DAP concentrations and GDS DQ scores at 12 and 24 months of age. DAPs

Infant (12 months) Motor domain Adaptive domain Language domain Social domain Child (24 months) Motor domain Adaptive domain Language domain Social domain

DMs

DEs

n

βa⁎ (95% CI)

p

βa (95% CI)

p

βa (95% CI)

297 297 297 297

0.14 (− 2.13, 2.41) −0.41 (−2.06, 1.23) 0.36 (− 1.32, 2.05) 0.67 (− 1.12, 2.45)

0.903 0.622 0.671 0.463

0.11 (−1.77, 1.99) −0.62 (− 1.98, 0.74) −0.21 (− 1.61, 1.19) −0.53 (− 2.00, 0.95)

0.910 0.371 0.765 0.484

0.46 0.27 0.57 1.48

262 262 262 262

−1.45 (−3.50, 0.60) −0.97 (−3.11, 1.17) 0.03 (− 2.14, 2.20) −2.49 (−4.85, −0.14)

0.166 0.372 0.978 0.038⁎

−0.75 −0.41 −0.61 −0.97

0.390 0.654 0.506 0.334

− 0.81 (− 2.66, 1.04) − 0.71 (− 2.63, 1.22) 0.31 (−1.65, 2.27) − 2.59 (− 4.71, − 0.46)

(− 2.46, (− 2.19, (− 2.43, (− 2.95,

0.97) 1.38) 1.20) 1.01)

(−1.58, (−1.21, (−0.94, (−0.12,

p 2.50) 1.75) 2.08) 3.08)

0.657 0.716 0.460 0.070 0.389 0.471 0.755 0.017⁎

DAPs, DMs, and DEs were log10-transformed. CI: confidence interval. a The model included maternal age, maternal IQ, maternal education, paternal education, household monthly income, smoking during pregnancy, and child gender. ⁎ p < 0.05.

measurements (e.g., cord blood vs. maternal urine), ethnicity/race, and sample size might account for some of the inconsistent results. In our study, only boys showed adverse association of prenatal exposure to OPs with neurodevelopment at 24 months of age, suggesting that the effect of OPs may differ by gender. Likewise, Liu et al. (2016) found that the negative relationship between prenatal exposure to OPs and neurodevelopmental delay at 24 months of age only existed in boys. The differences in hormone and repair procedures of damage, as well as the metabolism of OPs between males and females might account for the differential susceptibility to OPs by gender (Liu et al., 2016). Compared with females, males could produce more oxygen analogs and had a higher rate of hepatic activation of OPs (Sultatos, 1991), and the inhibition of cholinesterase was more substantially induced by OP exposure (Dam et al., 2000).

are in process, the adverse effect on neurodevelopment induced by neurotoxic compound exposure in an important period of development might appear later in time. The cascading developmental procedures start in the gestation period and go on in the first stages of infancy, explaining why the adverse effect on neurodevelopment could be detected at 24 months and not at an earlier age (Rice and Barone, 2000). In addition, some neurodevelopmental assessment methods are only sensitive enough for advanced children to exhibit adverse effects, which also could contribute to the delay between OP exposure and adverse effects (González-Alzaga et al., 2014). Limited epidemiological studies have examined the effects of prenatal exposure to OPs on childhood neurodevelopment, with inconsistent results. Researchers from the Jiangsu cohort found that prenatal exposure to DEs was related to neurodevelopmental delay in the adaptive domain at 24 months of age (n = 310) (Liu et al., 2016). Researchers from the CHAMACOS cohort investigated the association of prenatal exposure to OPs with neurodevelopment in 6-, 12-, and 24month-old children on the Bayley Scales of Infant Development and observed that children's MDI at 24 months (n = 372), rather than at 12 (n = 395) and 6 months (n = 396), was inversely associated with prenatal exposure to DMs instead of DEs or 3, 5, 6-trichloro-2-pyridinol (TCPy, a metabolite of chlorpyrifos and chlorpyrifos-methyl), and they found no relationships of prenatal OP exposure and PDI at any of the three time points (Eskenazi et al., 2007). However, researchers from the New York City cohort reported that prenatal blood concentrations of chlorpyrifos, which metabolizes into DEs, were responsible for the observed inverse relation with PDI and MDI in children aged 36 months instead of 12 and 24 months (n = 254) (Rauh et al., 2006). It should be noted that the differences in the neurodevelopmental tests, exposure

4.3. Associations between postnatal DAP concentrations and GDS DQ scores at 12 and 24 months of age In contrast to the results of prenatal exposure to OPs, we found that postnatal exposure to DMs and DAPs was positively related to adaptive domain DQ scores in 24-month-old children rather than in 12-monthold infants. Similar to our results, Eskenazi et al. (2007) observed that postnatal exposure to DMs and DAPs was positively associated with MDI at 24 months of age (n = 372), but no associations were observed between postnatal exposure to OPs and MDI or PDI at 12 months of age. Bouchard et al. (2011) found that DAPs at 12 months of age were associated with better scores on Verbal Comprehension and Full-Scale IQ based on the WICS-IV at 7 years of age (Wechsler Intelligence Scale for Children, 4th edition) (n = 329). One possibility is that children who

Table 4 Association between child DAP concentrations and GDS DQ scores at 12 and 24 months of age. DAPs

Infant (12 months) Motor domain Adaptive domain Language domain Social domain Child (24 months) Motor domain Adaptive domain Language domain Social domain

DMs

n

βa⁎ (95% CI)

235 235 235 235

0.75 0.26 1.57 0.66

(− 1.98, (− 1.74, (− 0.45, (− 1.51,

237 237 237 237

0.71 2.99 1.46 0.99

(− 1.59, 3.00) (0.58, 5.40) (− 1.07, 3.99) (− 1.75, 3.73)

3.48) 2.27) 3.59) 2.82)

DEs

p

βa (95% CI)

0.589 0.796 0.128 0.550

0.95 0.51 1.51 1.12

(− 1.40, (− 1.22, (− 0.23, (− 0.74,

0.546 0.015⁎ 0.256 0.476

0.84 3.00 2.10 0.27

(− 1.10, 2.78) (0.97, 5.04) (− 0.03, 4.23) (− 2.05, 2.59)

3.29) 2.24) 3.25) 2.98)

p

βa (95% CI)

p

0.428 0.562 0.088 0.235

0.15 (− 2.67, 2.96) −0.57 (−2.64, 1.50) −0.05 (−2.14, 2.04) −0.74 (−2.97, 1.49)

0.918 0.588 0.964 0.512

0.396 0.004⁎ 0.053 0.817

0.93 0.80 0.23 2.26

0.397 0.494 0.850 0.084

(− 1.23, (− 1.50, (− 2.15, (− 0.31,

3.09) 3.11) 2.61) 4.82)

DAPs, DMs, and DEs were log10-transformed. CI: confidence interval. a The model included maternal age, maternal IQ, maternal education, paternal education, household monthly income, smoking during pregnancy, and child gender. ⁎ p < 0.05.

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of urine samples collected throughout pregnancy may provide a more accurate estimate of the average exposure during the whole gestation. Fourth, in the present study, creatinine concentrations both in pregnant women (0.02–2.57 g/L) and in children (0.01–1.58 g/L) were relatively low, which might affect the reliability of the results. The widely accepted urinary creatinine level range is 0.3–3 g/L for occupational adults (WHO, 1996). We lack the reference value of urinary creatinine levels of Chinese pregnant women and children, especially young children aged 12–24 months. However, because urinary creatinine level varies by race/ethnicity, sex, age, body mass index, fat-free mass, and physiologic status (Barr et al., 2005; Eskenazi et al., 2007), the WHO standards may not be appropriate for our study populations who are undergoing rapid physiological changes, including pregnant women and young children. Therefore, we re-analyzed the association in two ways: We excluded samples with urinary creatinine levels below 0.05 g/L (Eskenazi et al., 2004; Wolff et al., 2008) and used log-transformed, non-creatinine-adjusted values (Liu et al., 2016). Interestingly, neither of them markedly altered the observed associations (data not shown). Fifth, other chemicals (i.e., pyrethroid pesticides, lead, mercury, and perfluoroalkyl and polyfluoroalkyl substances) were measured in most of the samples, but not all, due to the limited sample volume; hence, we were unable to include these suspected pollutants as confounders in the models. However, we tested collinearity between prenatal OP exposure and other pollutants by using the VIF in the model. All VIFs were < 5 and in an acceptable range, indicating that the effects of OPs may not be attributable to the effects of other pollutants.

had higher DQ scores were more likely to explore the environment more often, such as crawling and playing on the floor as well as putting things into their mouth, resulting in higher OP residue exposure (Eskenazi et al., 2007). However, Guodong et al. (2012) found no association between child urinary levels of OP metabolites and any of the DQ scores at 23–25 months of age (n = 301). Liu et al. (2016) found postnatal DEs and DAPs were related to neurodevelopmental delay in the social and motor domains at 24 months of age (n = 310). Thus, it would be challenging to assess the association between postnatal exposure to OPs and neurodevelopmental outcome. The differences in neurodevelopmental assessment methods, sample size, and exposure measure or scenario might account for the disparities of these inconsistent findings. The association of postnatal OP exposure and neurodevelopment were different from those of prenatal exposure to OPs, which might be explained by neuroplasticity. The nervous system could adapt to environmental toxins exposure and compensate for the adverse impacts as shown in several animal studies (Rice and Barone, 2000; Selemon, 2013). Furthermore, the neuroplasticity could be observed in humans, when a gradual maturation of fiber pathways, presumably supporting motor and speech functions, was found in adolescents and children (Paus et al., 1999). Our study contributes to the growing evidence that prenatal and postnatal exposure to OPs could affect children's neurodevelopment. Our study has a number of strengths. First, OP exposure was assessed not only by a questionnaire but also by analysis of urinary DAP metabolites in pregnant women and 12- and 24-month-old children (better than just a questionnaire survey). Second, to evaluate the effects of exposure to OPs on children comprehensively, we considered three critical windows of life, including prenatal and postnatal OP exposure at 12 and 24 months of age. We ran analyses to investigate prenatal and postnatal exposure in regression analyses together and found that the inverse relationship of prenatal OP exposure and DQ scores in the social domain in 24-month-old children still existed, and so did the positive association between postnatal exposure to OP and adaptive domain DQ scores in 24-month-old children (data not shown).Few studies have assessed the associations of not only prenatal but also postnatal exposure to OPs on childhood neurodevelopment based on a prospective birth cohort study in China, except for one study (Liu et al., 2016); thus, our research is of great significance, especially when the OP exposure level is high in China. Our study also has some limitations. First, our sample size was small, which may lead to less stable estimates of effect. Second, although the measurement of DAP metabolites is the most current technique to characterize and integrate multiple OP exposure from different sources, it should be noted that urinary metabolite levels may reflect exposure not only to OP parent compounds but also to the potentially less toxic preformed DAPs decomposed from the parent OPs in the environment (Lu et al., 2005). Third, only one spot urine specimen was used to analyze the level of DAP metabolites. It might be less representative of DAP concentrations in single spot urine samples to characterize exposure over a longer period of time, given the short halflives and high intra-individual variability inherent in these biomarkers of exposure (Bradman et al., 2013; Spaan et al., 2015). However, it was reported that a person's exposure level would be stable if they are in the same microenvironments for the same amount of time every day over the course of months and if levels of the contaminant in the microenvironments remain consistent over the same period (Meeker et al., 2005). For example, in the CHAMACOS cohort, two pregnancy total DAP concentrations [mean = 14.0 weeks gestation (GM: 113.5 nmol/ L) and 26.6 weeks gestation (GM: 116.9 nmol/L)] were very similar (Eskenazi et al., 2007). Therefore, we might speculate that the DAP concentrations in this study would likely have modest intra-individual variability because of the habitual lifestyle and the same microenvironments during pregnancy that suggest a constant exposure over days or months. Nevertheless, it is possible that repeated measurements

5. Conclusion We found that almost all the DAP concentrations in our study were higher than those reported in developed countries, and there was an inverse association between prenatal exposure to DEs and DAPs and DQs in social domain in children at 24 months of age. We also found that postnatal exposure to DMs and DAPs was positively associated with DQs in adaptive domain at 24 months of age. Our findings provide some evidence that OPs may affect neurodevelopment. Nevertheless, a large longitudinal study is required to explain the association and potential mechanisms linking OP exposure and neurodevelopment. Funding This work was supported by National Natural Science Foundation of China (Grant No. 81630085, 81602823, and 81402645), National Key Research and Development Program (2016YFC1000203), the National Basic Research Program of China (973 Program 2014CB943300), the Science and Technology Commission of Shanghai Municipality (17ZR1415800). Disclosures The authors declare no competing financial interest. Acknowledgements We thank the Department of Environmental Health staff, students, participants, and hospital partners. We specifically thank Weituo Zhang for his work on statistical guidance. Appendix A. Supplementary data Additional information is included in this study: detailed description of method to quantify the selected DAPs; on accuracy, precision, LOD, and LOQ of analytical procedure (Supplementary Table 1); distribution of GDS DQ scores of children at 12 and 24 months of age (Supplementary Table 2); association between maternal DAP 125

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