Exposure to carbamate and neurodevelopment in children: Evidence from the SMBCS cohort in China

Environmental Research 177 (2019) 108590

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Exposure to carbamate and neurodevelopment in children: Evidence from the SMBCS cohort in China☆,☆☆

T

Jiming Zhanga, Jianqiu Guoa, Chunhua Wua, Xiaojuan Qia,b, Shuai Jianga, Dasheng Luc, Chao Fengc, Weijiu Liangd, Xiuli Changa, Yubin Zhanga, Yang Caoe, Guoquan Wangc, Zhijun Zhoua,* a

School of Public Health, Key Laboratory of Public Health Safety of Ministry of Education, Collaborative Innovation Center of Social Risks Governance in Health, Fudan University, No.130 Dong'an Road, Shanghai, 200032, China b Zhejiang Provincial Center for Disease Control and Prevention, No.3399 Binsheng Road, Hangzhou, 310051, China c Shanghai Center for Disease Control and Prevention, No.1380 West Zhongshan Road, Changning District, Shanghai, 200336, China d Changning Center for Disease Control and Prevention, No.39 Yunwushan Road, Changning District, Shanghai, 200051, China e Clinical Epidemiology and Biostatistics, School of Medical Sciences, Örebro University, Örebro, 70182, Sweden

A R T I C LE I N FO

A B S T R A C T

Keywords: Carbamate pesticide Carbofuranphenol Prenatal exposure Postnatal exposure Neurodevelopment

Background: Carbamate pesticides exposure have been linked with adverse health effects during developmental period. Based on 377 mother-child pairs from Sheyang Mini Birth Cohort Study, the present study aimed to assess carbofuranphenol exposure of three-year-old children and explore the associations between prenatal or postnatal carbofuranphenol exposures and neurodevelopmental indicators. Methods: Urinary carbofuranphenol concentrations were measured by gas chromatography-tandem mass spectrometry. Neural developmental quotient (DQ) of children was evaluated using Gesell Developmental Schedules. Generalized linear models were used to examine the associations between carbofuranphenol concentrations and neurodevelopment. Results: Geometric mean, geometric standard deviation, median, inter quartile range of postnatal urinary carbofuranphenol concentrations were 0.653 μg/L, 9.345 μg/L, 0.413 μg/L, 0.150–1.675 μg/L, respectively. Postnatal carbofuranphenol level showed negatively significant trend in language DQ [beta (β) = -0.121; 95% confidence interval (95% CI): 0.212, −0.031; p value (p) = 0.008] and total average DQ (β = −0.059, 95% CI: 0.115, −0.003; p = 0.035). Prenatal carbofuranphenol level showed negative correlations with children's adaptive DQ (β = −0.755; 95% CI: 1.257, −0.254; p = 0.003), social DQ (β = −0.341; 95% CI: 0.656, −0.027; p = 0.032) and total average DQ (β = −0.349; 95% CI: 0.693, −0.005; p = 0.047). Conclusion: The results of the present study supposed children in agricultural region of China are widely exposed to carbamate pesticides, and both prenatal and postnatal exposure to carbamate pesticides may lead to neurodevelopmental effect.

1. Introduction

pesticides due to potentially ecological and health effects since 2006 (Environmental Protection Agency, 2006; European Food Safety Authority, 2015), they are still used in other countries (Center of Disease Control and Prevention, 2016). China has banned the use of carbofuran, benfuracarb and carbosulfan in vegetable, fruits, tea and

Carbamate pesticides, such as carbofuran, benfuracarb, and carbosulfan are widely used in agricultural pest control. Although European countries and the United States (U.S.) have restricted the use of these

☆ This study was jointly supported by the Joint China-Sweden Mobility Program of the Swedish Foundation for International Cooperation in Research and Higher Education (STINT CH2015-6145) and the Shanghai 3-Year Action Project (GWIV-27.3 & 15GWZK0201). ☆☆ The study protocol was approved by the Ethics Committee of the School of Public Health, Fudan University. All the participants or caregivers have signed informed consents. * Corresponding author. E-mail addresses: [email protected] (J. Zhang), [email protected] (J. Guo), [email protected] (C. Wu), [email protected] (X. Qi), [email protected] (S. Jiang), [email protected] (D. Lu), [email protected] (C. Feng), [email protected] (W. Liang), [email protected] (X. Chang), [email protected] (Y. Zhang), [email protected] (Y. Cao), [email protected] (G. Wang), [email protected] (Z. Zhou).

https://doi.org/10.1016/j.envres.2019.108590 Received 11 April 2019; Received in revised form 11 July 2019; Accepted 15 July 2019 Available online 16 July 2019 0013-9351/ © 2019 Elsevier Inc. All rights reserved.

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prevent plant diseases. Carbofuran is one of the most extensive applied pesticides in Sheyang (Qin et al., 2007). SMBCS is a prospective hospital-based cohort study conducted in Sheyang County, which focuses on environmental pollutant exposures during prenatal and postnatal periods and development in children. During June 2009 to January 2010, a total of 1303 healthy pregnant women residing in Sheyang County over one year volunteered to participate in the Sheyang Mini Birth Cohort Study and donated their urine samples at the ward of Sheyang Maternity Hospital (Qi et al., 2012). 203 mother-child pairs were excluded due to stillbirth (1 pair), congenital anomalies (9 pairs), multiparity (9 pairs), incomplete data (30 pairs) or lack of urine sample (154 pairs). A total of 1100 mother-child pairs were included in the study of prenatal carbofuranphenol level and birth outcomes (Zhang et al., 2018). 377 of the 1100 pairs completed neurodevelopment assessment and survey questionnaire, and donated child's urine sample. The included 377 pairs were not statistically different from the initial participants regarding sociodemographic features and other key covariates (Supplemental material 2).

Chinese herb planting, but they can still be legally applied on cereal crop and cotton (China Pesticide Information Network, 2016). In China, the application of carbofuran in 2015 was about 12,438 tons (China Pesticide Web, 2017), and environmental monitoring confirmed the presence of carbofuran and its derived pesticides (benfuracarb and carbosulfan) in groundwater, soils, and agricultural products (Fang et al., 2015; Geng et al., 2017; Xu et al., 2016; Zhang et al., 2016). Body burden of carbamate pesticides in general population is mainly sourced from the consumption of contaminated food (Forde et al., 2015). In vivo, carbofuran, benfuracarb and carbosulfan are metabolized into carbofuranphenol (2, 3-dihydro-2, 2-dimethyl-7-hydroxybenzofuran) and 3-ketocarbofuran that are eliminated in urine rapidly. For example, metabolite half-life of carbofuran in rat is 29 ± 5 min and the elimination rates after 6 h, 12 h, 24 h, 48 h are 21.4%, 37.8%, 72.2%, 87.7%, respectively (Dorough, 1968; Ferguson et al., 1984). Since carbofuranphenol is a main degradation metabolite of carbamates, urinary carbofuranphenol level is regarded as a biomarker to evaluate carbamate exposure in epidemiological studies (Castorina et al., 2010; Center of Disease Control and Prevention, 2017; Forde et al., 2015). Carbofuran is not only an acetylcholinesterase inhibitor but also a recognized endocrine disruptor (Cranmer et al., 1978). Toxicological experiments have shown that carbofuran can cross placental barrier and lead to nonspecific developmental effects, such as reduced weight gain, shorter survival, testicular toxicity, hormones unbalance, and affect fetal development in utero (Baligar and Kaliwal, 2002; Goad et al., 2004; Mishra et al., 2012; Pant et al., 1995, 1997). Fu et al. (2019) reported synergistic neurotoxicity of carbofuran on SK-N-SH cells, and the toxicity remained even in low-dose condition. Time-course and dose-response data documented greater sensitivities for carbofuran in young rats (Moser et al., 2010). The endocrine disrupt effect of carbamate may have low-dose effects leading reverse effect at very low dose (Vandengberg, 2014), the potential adverse health effects of long-term low-level exposure to carbamates have attracted attention. According to the Developmental Origin of Health and Diseases (DOHaD) theory, health status in childhood may affect health status in whole life (Barker, 1999), and neurodevelopmental disorders may cause lifelong disability (Grandjean and Landrigan, 2014). Several children-based studies on acetylcholinesterase inhibitory pesticides were published in recent years, the inconsistent results including positive effects (Grandjean et al., 2006; Harari et al., 2010), negative effects (Kofman et al., 2006; Liu et al., 2016) and none effects (Bouchard et al., 2011; Ding et al., 2012) have caused increasing concern of developmental effects. Nevertheless, little is known about carbamate burden in young children and the neurodevelopmental effects of carbamate exposure in early life from population-based study. In the current study, we quantified the body burden of carbofuranphenol in 3-year-old children from the Sheyang Mini Birth Cohort Study (SMBCS) and explored the associations between prenatal or postnatal carbofuranphenol exposures and children's neurodevelopment. It was hoping to contribute scientific data for policy making on pesticide control and provide population-based evidence for non-occupational risk assessment not only in China but also internationally. The SMBCS protocols for both the parent study and current study were approved by the Ethics Committee of the School of Public Health, Fudan University. All the participants or caregivers have signed informed consents.

2.2. Data and urine sample collection Face-to-face interviews for pregnant women and children were conducted to capture information on lifestyles, socioeconomic status, and environmental characteristics (Qi et al., 2012; Guo et al., 2017). The interviewers were well trained and blinded to carbofuranphenol exposure and neurodevelopmental status of children. Prenatal spot urine sample was obtained from each mother prior to delivery in the ward, mean and standard deviation of prenatal sample collection time were 39.40 ± 1.07 weeks. Postnatal urine samples were collected when the children were followed up at Sheyang Health Care Center, and a neurodevelopmental test was completed at the same day. The age (mean ± standard deviation) of the children at the end of 3-year follow-up was 37.54 ± 2.37 months. The urine samples were immediately transferred to the high-density polypropylene centrifuge tubes (Corning Incorporated, USA) and stored at −20 °C, then were long-term kept frozen at −80 °C until analysis. 2.3. Urine carbofuranphenol analysis The determination method for carbofuranphenol in urine using gas chromatography-tandem mass spectrometry (GC-MS/MS) was detailly described in our previous study (Lu et al., 2015). Briefly, 0.5 mL urinary sample was hydrolyzed by 125 μL hydrochloric acid, followed by continuous extraction twice with 2 mL of a mixture of hexane and methyl tert-butyl ether (1:1, v/v). Then the extracted sample was cleaned up using K2CO3-treated-silica-gel solid phase extraction (SPE) cartridge. After derivatization with N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA), the carbofuranphenol was measured in maternal and children's urine by GC-MS/MS. The average recovery of carbofuranphenol was nearly 100% and the limit of detection (LOD) for carbofuranphenol was 4.4 ng/L, range of quantitation was 0.1–100 μg/mL, and 5% samples were duplicate detected. 2.4. Neurodevelopmental assessment Neurodevelopmental outcomes were assessed with the Gesell Developmental Schedules (GDS, Liu et al., 2016). The GDS was adapted to the Chinese population after revision (Beijing Mental Development Cooperative Group, 1985), which is well-used in China to assess neurodevelopment during early childhood (Ding et al., 2012; Wang et al., 2017). GDS was performed by trained pediatricians using standardized procedures, and the pediatricians were blinded to exposure and sociodemographic status. Four main neurobehavioral functions were derived from items in GDS. Briefly, motor function, including locomotion, reaching, balance, comprehension, drawing, hand control; adaptive behavior, including

2. Method 2.1. Study population Sheyang County of Jiangsu Province (Supplemental Material 1) located in East China (N 33°46′, E 120°15′), has a Yellow Sea coastline of 103 km. It is well known for agricultural products, such as rice and cotton, thus, a great deal of pesticides is applied to control pests and 2

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eye-hand coordination, imitation, object recovery, discriminative performance, perception, completion, number conception; language behavior assessed by means of vocabulary, word comprehension, conversation, word production; social behavior, including reactions to persons, personal habits, initiative and independence, play responses, acquired information (Tang et al., 2008). The pediatricians estimated a maturity age of neurodevelopmental status (expressed in months) based on children's performance. Each child was assessed by one pediatrician for the specific area, and was assigned a developmental quotient (DQ) accordingly, and the total average quotient was calculated as mean of four DQs. To minimize both inter-examiner and intra-examiner variability and maximize reliability in scoring, standardized training procedures and regular self-checking were performed throughout the assessment.

Table 1 Characteristics of participants [n (%) or mean ± standard deviation]. Included (n = 377) Maternal age at birth Gestational week Education level Middle school or lower High school or higher Family annual income < 30,000 RMB ≥30,000 RMB Family member smokes at home Yes No Neonatal gender Male Female Inhabitation type Town Suburb Countryside Housing to agricultural land less than 100m Housing to urban green land less than 100m Paternal occupation Agricultural worker Industrial Government and public institution employee Others Unemployed Maternal occupation Agricultural worker Industrial Government and public institution employee Others Unemployed

2.5. Statistical analysis Differences in socio-demographic characteristics and maternal carbofuranphenol concentrations between included mother-child pairs in the present study and original subjects were tested using chi-squared test (for categorical variables) and Mann-Whitney U test (for continuous variables). Mann-Whitney U test was also used to examine the difference in distributions of genders, follow-up seasons and family locations. Correlations between maternal and childhood urinary carbofuranphenol levels were assessed using Pearson correlation coefficients. Prenatal carbofuranphenol level was adjusted for creatinine to account for urinary dilution factor. Generalized linear models (GLM) were performed to examine the associations between the prenatal or postnatal carbofuranphenol levels and DQ scores. Prenatal and postnatal carbofuranphenol concentrations were divided into quartiles, the 1st (lowest) concentration quartiles were set as reference. And, each quartile was presented using its median value (0.134, 0.530, 1.297, 4.766 for prenatal; 0.074, 0.269, 0.746, 18.85 for postnatal) to explore p-trend value in GLMs. Log-transformed urinary creatinine concentrations were added into GLMs to adjust dilution effect. Potential confounding variables were known or suspected risk factors for the carbofuranphenol exposure or neurodevelopment based on our previous studies and literatures (Barr et al., 2010; Guo et al., 2016; Qi et al., 2012). Variables were included as covariates in the multivariate regression models if they were related to carbofuranphenol exposure and children neurodevelopment (p < 0.10), or changes the coefficient of urinary carbofuranphenol concentration by more than 10%. Finally, the following covariates were included: maternal age at birth, education level (middle school or lower, high school or higher), annual household income (over 30,000 RMB or not), family location (town, suburb or countryside), passive smoking (yes or not), follow-up season of postnatal sample (spring for February to April, summer for May to July, autumn for August to October, winter for November to January). An interaction term between sex and carbofuranphenol level was included in each GLM, further analyses stratified by children's sex would be conducted when the interaction term with a P value lower than the given cut-off 0.10. 3,5,6-trichloro-2-pyridinol (both prenatal and postnatal, specific metabolite of Chlorpyrifos, TCPY) and 3Phenoxybenzoic acid (prenatal only, nonspecific metabolite of pyrethroid pesticides, 3-PBA) were selected as sensitivity parameters to exam the robustness of the model and explore unexpected relationship with other kinds of pesticide. All data were entered into Epidata 3.1 software (The Epidata Association, Odense, Denmark). The statistical analyses were performed using SAS version 9.4 (SAS Institute, Cary, NC) and IBM SPSS 18.0 for Windows (IBM Corporation, Armonk, New York). A two-sided P value < 0.05 was considered as statistically significant.

25.92 ± 5.59 39.40 ± 1.07 265 (70.3%) 112 (29.7%) 169 (44.8%) 208 (55.2%) 173 (45.9%) 204 (54.1%) 184 (48.8%) 193 (51.2%) 130 (34.6%) 129 (34.0%) 118 (31.4%) 98 (26.0%) 78 (20.7%) 8 (2.1%) 105 (27.9%) 37 (9.8%) 219 (58.1%) 8 (2.1%) 4 (1.1%) 77 (20.4%) 38 (10.1%) 118 (31.3%) 140 (37.1%)

3. Results 3.1. Characteristics of participants In current study, average maternal age at delivery was 25.9 years; 29.7% of the mothers completed high school or higher education; over half (55.2%) of the participating families had an annual household income over 30,000 RMB during pregnancy; 51.2% of the children were female; 45.9% of the children lived with smoker; inhabitation types of town (34.6%), suburb (34.0%), and countryside (31.4%) were roughly even. 8 fathers and 4 mothers were agricultural worker, 98 children's inhabitations were less than 100 m from agricultural land. In terms of these characteristics, participants included in current study were not statistically different from those enrolled initially (Table 1), except for education level (p = 0.023). The mean ± standard deviations (SDs) of the motor, adaptive, language, social, and total average DQ were 94.7 ± 6.0, 92.3 ± 7.7, 93.3 ± 8.3, 93.3 ± 6.1, and 93.3 ± 6.2, respectively.

3.2. Urinary carbofuranphenol levels Maternal urinary carbofuranphenol levels had been reported previously (Zhang et al., 2018). Carbofuranphenol was detected in 365 of 377 (96.8%) urine samples. Geometric mean, geometric standard deviation, median, inter quartile range of postnatal urinary carbofuranphenol concentrations were 0.653 μg/L, 9.345 μg/L, 0.413 μg/L, 0.150–1.675 μg/L, respectively. The statistics by subgroups were shown in Table 2. Carbofuranphenol concentrations were not statistically different between genders, seasons, and family locations.

3

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Table 2 Urinary carbofuranphenol levels of 3-year-old children from SMBCS. n Total Gender Male Female Season Spring Summer Autumn Winter Family location Town Suburb Countryside

%

377

GM

GSD

Min

P25

Median (P50)

P75

Max

0.653

9.345

< LOD

0.150

0.413

1.675

162.791

184 193

48.8 51.2

0.677 0.632

9.867 8.910

< LOD < LOD

0.150 0.150

0.449 0.381

2.082 1.595

162.791 159.378

61 68 201 47

16.2 18.0 53.3 12.5

0.692 0.964 0.630 0.404

10.009 9.651 9.242 8.237

< LOD < LOD < LOD < LOD

0.155 0.275 0.139 0.135

0.443 0.778 0.403 0.263

3.138 2.790 1.547 0.768

106.305 159.378 162.791 63.234

130 129 118

34.5 34.2 31.3

0.555 0.778 0.694

10.030 8.997 8.997

< LOD < LOD < LOD

0.149 0.188 0.143

0.426 0.591 0.395

1.347 1.613 2.335

109.409 162.791 159.378

GM: Geometric Mean; GSD: Geometric Standard Deviation; P25: 25th Percentile; P50: 50th Percentile; P75: 75th Percentile.

−0.031; p = 0.044) than the reference quartile. (Table 3).

3.3. Association between postnatal carbofuranphenol levels and neurodevelopment

3.4. Association between prenatal carbofuranphenol levels and neurodevelopment

All GLMs passed the omnibus test with P values less than 0.001 and the residuals were normally distributed (Kolmogorov-Smirnov Test). Results derived from the GLMs on postnatal carbofuranphenol levels and DQs were presented in Table 3. Postnatal carbofuranphenol level showed negatively significant trend in language DQ [beta (β) = -0.121; 95% confidence interval (95% CI): 0.212, −0.031; p value (p) = 0.008] and total average DQ (β = −0.059, 95% CI: 0.115, −0.003; p = 0.035). P value of all the sex-interaction terms were over the cutoff. The children with highest postnatal exposure in the 4th quartile performed worse in language DQ (β = −2.492, 95% CI: 4.520, −0.463; p = 0.016) and social DQ (β = −1.160, 95% CI: 2.288,

Prenatal GLMs showed negative correlations between maternal urinary carbofuranphenol level and children's adaptive DQ (β = −0.755; 95% CI: 1.257, −0.254; p = 0.003), social DQ (β = −0.341; 95% CI: 0.656, −0.027; p = 0.032) and total average DQ (β = −0.349; 95% CI: 0.693, −0.005; p = 0.047). P values of sex-interaction terms for adaptive DQ and total average DQ were under 0.10. Statistical decrease trend in adaptive DQ was observed in female children (β = −0.693; 95% CI: 1.326, −0.059; p = 0.032). The children in the 2nd quartile (β = 1.212; 95% CI: 0.170, 2.253; p = 0.023) and 3rd quartile (β = 1.295; 95% CI: 0.253, 2.336; p = 0.015) had higher motor DQs, compared with the 1st quartiles (Table 4).

Table 3 Results of generalized linear models of postnatal carbofuranphenol levels and DQs. beta Motor DQ

Adaptive DQ

Language DQ

Social DQ

Total average DQ

1st quartile 2nd quartile 3rd quartile 4th quartile Sex-interaction p-trend 1st quartile 2nd quartile 3rd quartile 4th quartile Sex-interaction p-trend 1st quartile 2nd quartile 3rd quartile 4th quartile Sex-interaction p-trend 1st quartile 2nd quartile 3rd quartile 4th quartile Sex-interaction p-trend 1st quartile 2nd quartile 3rd quartile 4th quartile Sex-interaction p-trend

Ref. −0.187 0.878 −0.394 −0.009 −0.031 Ref. −1.153 0.184 −1.043 −0.069 −0.042 Ref. −0.305 0.061 −2.492 −0.146 −0.121 Ref. −0.739 0.039 −1.160 −0.026 −0.050 Ref. −0.293 0.546 −1.021 −0.073 −0.059

95% CI

3.5. Sensitivity analysis

p

(-1.236, (-0.172, (-1.440, (-0.102, (-0.078,

0.863) 1.929) 0.653) 0.083) 0.015)

0.728 0.101 0.461 0.842 0.192

(-2.982, (-1.648, (-2.867, (-0.231, (-0.124,

0.677) 2.015) 0.780) 0.092) 0.039)

0.217 0.844 0.262 0.398 0.307

(-2.343, (-1.977, (-4.520, (-0.324, (-0.212,

1.733) 2.099) −0.463) 0.031) −0.031)

0.769 0.953 0.016 0.105 0.008

(-1.871, (-1.094, (-2.288, (-0.126, (-0.100,

0.394) 1.173) −0.031) 0.073) 0.000)

0.201 0.946 0.044 0.597 0.052

(-1.532, (-0.694, (-2.256, (-0.182, (-0.115,

0.946) 1.787) 0.213) 0.035) −0.003)

0.643 0.388 0.105 0.185 0.035

The results derived from the models containing TCPY and 3-PBA were almost the same as above (Supplemental material 3). 4. Discussion The present study aimed to evaluate the effects of prenatal and postnatal carbamate exposure on neurodevelopment in children living in an agricultural region in Eastern China. We found that the 3-year-old children in the SMBCS were widely exposed to carbamate pesticides. Several statistically significant associations between carbofuranphenol levels and neurodevelopmental indicators suggested negative neurodevelopmental effect of carbamate exposure. With the improved detection method, carbofuranphenol was detected in 96.8% of postnatal urine samples, that built the basement of subsequent effect explorations. To our best knowledge, only the National Health and Nutrition Examination Surveys (NHANES) reported urinary carbofuranphenol concentrations of children aged 6–11. With a LOD of 0.4 μg/L, the 95th percentile of NHANES 99-00 is 0.570 μg/L, and carbofuranphenol was not detected in all samples of NHANES 01–02 (Center of Disease Control and Prevention, 2017), which means the concentrations in all samples were lower than 0.4 μg/ L. Comparing with the results of NHANES, the median concentration of the current study (0.413 μg/L) was quite similar to the LOD of NHANES, and the 95th percentile (64.421 μg/L) was much higher than the 95th percentile of NHANES 99-00, that supposed the exposure level of children from the SMBCS is higher than that of the NHANSE children. The results of GLMs showed negative associations between carbofuranphenol levels and adaptive DQ, language DQ, social DQ and total

CI: Confidence Interval; DQ: developmental quotient. Covariates: maternal age at birth, education level, family annual income, family location, passive smoking, season of sample collection. 4

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Table 4 Results of generalized linear models of prenatal carbofuranphenol levels and DQ scores. Total beta Motor DQ

Adaptive DQ

Language DQ

Social DQ

Total average DQ

1st quartile 2nd quartile 3rd quartile 4th quartile Sex-interaction p-trend 1st quartile 2nd quartile 3rd quartile 4th quartile Sex-interaction p-trend 1st quartile 2nd quartile 3rd quartile 4th quartile Sex-interaction p-trend 1st quartile 2nd quartile 3rd quartile 4th quartile Sex-interaction p-trend 1st quartile 2nd quartile 3rd quartile 4th quartile Sex-interaction p-trend

Ref. 1.212 1.295 0.202 0.041 −0.125 Ref. 0.858 0.14 −0.319 0.961 −0.755 Ref. 1.669 1.765 −0.036 0.594 −0.380 Ref. 0.972 0.857 −0.745 0.311 −0.341 Ref. 0.913 1.006 −0.231 0.415 −0.349

Male 95% CI

p

(0.170, 2.253) (0.253, 2.336) (-0.848, 1.252) (-0.363, 0.446) (-0.417, 0.167)

0.023 0.015 0.706 0.840 0.401

(-0.968, 2.684) (-1.687, 1.966) (-2.160, 1.522) (0.267, 1.655) (-1.257, −0.254)

0.357 0.881 0.734 0.006 0.003

(-0.366, (-0.265, (-2.082, (-0.188, (-0.946,

3.704) 3.794) 2.010) 1.377) 0.185)

0.108 0.088 0.972 0.136 0.187

(-0.147, (-0.263, (-1.874, (-0.123, (-0.656,

2.092) 1.977) 0.384) 0.745) −0.027)

0.089 0.134 0.196 0.160 0.032

(-0.321, (-0.229, (-1.476, (-0.061, (-0.693,

2.148) 2.241) 1.014) 0.891) −0.005)

0.147 0.110 0.716 0.087 0.047

Female

beta

95% CI

p

beta

95% CI

p

Ref. 0.752 0.78 0.073

(-1.007, 2.512) (-0.943, 2.504) (-1.734, 1.880)

0.402 0.375 0.937

Ref. 1.195 −0.079 −0.644

(-1.968, 4.358) (-3.306, 3.148) (-3.774, 2.486)

0.459 0.962 0.687

0.136

(-0.213, 0.486)

0.445

−0.693

(-1.326, −0.059)

0.032

Ref. 0.752 0.78 0.073

(-1.007, 2.512) (-0.943, 2.504) (-1.734, 1.880)

0.402 0.375 0.937

Ref. 1.195 −0.079 −0.644

(-1.968, 4.358) (-3.306, 3.148) (-3.774, 2.486)

0.459 0.962 0.687

0.050

(-0.281, 0.382)

0.764

−0.358

(-0.729, 0.011)

0.057

CI: Confidence Interval; DQ: developmental Quotient. Covariates: maternal age at birth, education level, family annual income, family location, passive smoking, season of sample collection.

measured by increased reactive oxygen species and reactive nitrogen species generation, and high-energy phosphates depletion (Milatovic et al., 2005). Gupta et al. (2011) reported carbofuran exposure inducing a spectrum of deficits on neuro system in rodents, including increasing oxidative and nitrosative stress, alteration in energy metabolism and consequent degeneration of pyramidal neuron from hippocampal region. In the present study, sex-differential effects were observed. Organophosphate pesticides (González-Alzaga et al., 2015) and chemicals other than pesticides such as trace metals (Rodríguez-Barranco et al., 2014) and air pollutants (Clougherty, 2010) showed more pronounced neurotoxic effects in male children than female children. Carbamate pesticides also acted sex-differentially in animal experiments. Yi et al. (2006) reported that decarbamylation rate for carbofuran in carps (Carassius auratus) was different between genders. In view of differences in pesticides’ health effects between genders observed in several researches, more researches are needed to clarify the sex/gender-differential neurodevelopmental effects in humans. The present study is based on a prospective cohort study with several advantages. The sample size of current study is moderate and the exposure measurement technology is efficient. The exposure levels and neurodevelopmental effects in children in early life were assessed, which could partially fill up the knowledge gap in carbamate researches. Carbofuranphenol was detected in more than 95% of the children's urine samples that implies the children in agricultural region of China being widely exposed to carbamate pesticides, and the high detection rate makes it possible to explore the health effects. Several adverse effects of carbamate exposure were found, which suggest that carbamate may affect neurodevelopment in childhood. Since carbamate pesticides still can be legally applied on cereal crop and cotton, the main exposure source of children is contaminated agricultural products. However, children live near agricultural land

average DQ, suggested negative health effect of early-life carbamate exposure. There are three possible reasons of the neurodevelopmental effect. Firstly, developmental neurotoxicity of carbamate may affect development of central nervous system (Gupta et al., 2011). Gupta et al. (2011) reported carbofuran exposure inducing a spectrum of deficits on neuro system in rodents, including increasing oxidative and nitrosative stress, alteration in energy metabolism and consequent degeneration of pyramidal neuron from hippocampal region. The research of Mishra et al. (2012) suggested that early gestational carbofuran exposure diminishes neurogenesis, reduces the neuro precursor cell pool, produces neurodegeneration in the hippocampus, and causes cognitive impairments in rat offspring. Since learning, memory, cognitive functions, and hippocampus are closely related to language and social abilities of human, the significant associations in highest exposure groups indicate that carbamate exposure at current level may affect children's neurodevelopmental process. Seth et al. (2017) reported that both in vitro and in vivo studies indicated carbofuran's inhibitory effects on neurogenesis and associated learning and memory deficits, which suggest that carbofuran inhibits neural stem cell proliferation and neuronal differentiation by altering TGF-β signaling. Seth et al. (2019) also reported that both prenatal and postnatal exposure to carbofuran alters myelination potential in the hippocampus, which may lead to cognitive deficits in rats. Secondly, endocrine disrupt effect may be a cause of neurodevelopmental risk. Environmentally relevant doses of highly effective and potent signaling hormones may lead to non-directional physiological changes (Vandengberg, 2014). As an endocrine disruptor, exposure of carbofuran lead to progesterone, cortisol, estradiol increasing (Goad et al., 2004), and prenatal exposure to carbofuran may result in postnatal endocrine change or dysfunction (Cranmer et al., 1978). Thirdly, the general toxicity or developmental toxicity of carbamate pesticides may delay overall developmental process. Carbofuran-induced muscle hyperactivity produces oxidative stress as 5

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may exposure carbamates from groundwater or soils, children live with agricultural worker may touch contaminated clothes or tools. The potential neurobehavior effects of carbamates may increase children's hand-mouth behavior that reversely increases the exposure level. Nevertheless, there are several limitations exist in current study. Because of the short half-life of carbofuranphenol in body, the carbofuranphenol level measured only in one spot urine sample may cause exposure misclassification. Moreover, as we enrolled participants from only one maternity hospital, the selection bias may exist which could limit the generalization of the findings, thus, cross-cohort cooperation or multi-center research are needed to further confirm our findings.

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5. Conclusion The current research provided population-based evidence for carbamates’ risk management, and indicated that children in an agricultural region of China were widely exposed to carbamate, both prenatal and postnatal exposure to carbamate pesticides might affect neurodevelopmental process in childhood. Since contaminated agricultural products is a major source of carbamate exposure, the residue of carbamate should be including in regular quality monitoring, and the residue limitation should be set according to results of relative researches. Moreover, playing in agricultural land and touching contaminated clothes or tools of agricultural workers should also be avoided to protect children from potential health risk of carbamate. Further mechanism and epidemiological studies are needed to clarify the health effects of carbamate pesticides on neurodevelopment and the relationship between the exposure and behavioral problem. Conflicts of interest The authors declare that they have no actual or potential competing interests. Acknowledgment This study was jointly supported by the Joint China-Sweden Mobility Program of the Swedish Foundation for International Cooperation in Research and Higher Education (STINT CH2015-6145) and the Shanghai 3-Year Action Project (GWIV-27.3 & 15GWZK0201). We greatly appreciate the participating mother-child pairs in the SMBCS. We also acknowledge the cooperation of the colleagues in Sheyang County Maternity Hospital, Jiangsu Province (China) and Sheyang Maternal and Child Health Care Center, Jiangsu Province (China). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.envres.2019.108590. References Baligar, P.N., Kaliwal, B.B., 2002. Reproductive Toxicity of Carbofuran to the Female Mice: Effects on Estrous Cycle and Follicles. Industrial Health 40, 345–352. https:// doi.org/10.2486/indhealth.40.345. Barker, D.J.P., 1999. Fetal origins of cardiovascular disease. Ann. Med. 31 (Suppl. 1), 3–6. https://doi.org/10.1080/07853890.1999.11904392. Barr, D.B., Ananth, C.V., Yan, X., Lashley, S., Smulian, J.C., Ledoux, T.A., Hore, P., Robson, M.G., 2010. Pesticide concentrations in maternal and umbilical cord sera and their relation to birth outcomes in a population of pregnant women and newborns in New Jersey. Sci. Total Environ. 408, 790–795. https://doi.org/10.1016/j.scitotenv. 2009.10.007. Beijing Mental Development Cooperative Group, 1985. Gesell developmental diagnosis scale. Beijing Mental Development Cooperative Group. Bouchard, M.F., Chevrier, J., Harley, K.G., Kogut, K., Vedar, M., Calderon, N., Trujillo, C., Johnson, C., Bradman, A., Barr, D.B., Eskenazi, B., 2011. Prenatal exposure to organophosphate pesticides and IQ in 7-year-old children. Environ. Health Perspect. 119, 1189–1195. https://doi.org/10.1289/ehp.1003185.

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