Attachment_V_-_Sample_Task_Order.pdf

ATTACHMENT V – SAMPLE TASK ORDER

JOURNAL COPYEDITING & PAGE COMPOSITION

ENVIRONMENTAL HEALTH PERSPECTIVES

NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES

RESEARCH TRIANGLE PARK, NORTH CAROLINA

ITEM 1 - TEST MANUSCRIPT

Prenatal PBDE and PCB exposures and reading, cognition, and externalizing 1 behavior in children 2

Hongmei Zhang1, Kimberly Yolton2, Glenys M. Webster3, Andreas Sjödin4, Antonia M. Calafat4, 3

Kim N. Dietrich5, Yingying Xu2, Changchun Xie5, Joseph M. Braun6, Bruce P. Lanphear3, 4

Aimin Chen5 5

1Department of Environmental Health, School of Public Health, Shanxi Medical University, 7

Taiyuan, Shanxi Province, China; 2Division of General and Community Pediatrics, Department 8 of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA; 3Child and 9

Family Research Institute, BC Children’s and Women’s Hospital and Faculty of Health Sciences, 10

Simon Fraser University, Vancouver, British Columbia, Canada; 4Division of Laboratory 11

Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, 12

Atlanta, Georgia, USA; 5Department of Environmental Health, University of Cincinnati College 13 of Medicine, Cincinnati, Ohio, USA;6Department of Epidemiology, Brown University School of 14

Public Health, Providence, Rhode Island, USA. 15

Corresponding author: Dr. Aimin Chen, Division of Epidemiology, Department of 17

Environmental Health, University of Cincinnati College of Medicine, P.O. Box 670056, 18

Cincinnati, OH 45267-0056, USA. Tel: 513-558-2129, Fax: 513-558-4397, E-mail: 19 aimin.chen@uc.edu 20

Running title: Prenatal PBDE and PCB exposures and reading. 22

Acknowledgments 23

This work was supported by grants from the US National Institute of Environmental Health 24

Sciences and the Environmental Protection Agency (NIEHS P01 ES11261, R01 ES014575, R01 25

ES020349, and R00 ES020346; EPA P01R829389). The findings and conclusions in this report 26 are those of the authors and do not necessarily represent the official position of the NIEHS or the 27

Centers for Disease Control and Prevention (CDC). 28

Competing financial interest declaration 30

Dr. Lanphear has served as an expert witness and a consultant to the California Attorney 31

General’s Office for the plaintiffs in a public nuisance case related to childhood lead poisoning, 32 but he has not personally received any compensation for these services. Dr. Lanphear has also 33 served as a paid consultant on a US Environmental Protection Agency research study related to 34 childhood lead poisoning. Dr. Braun was financially compensated for conducting a re-analysis of 35 a study of child lead exposure for the plaintiffs in a public nuisance case related to childhood 36 lead poisoning. None of these activities are directly related to the present study. The other 37 authors declare they have no actual or potential competing financial interests. 38

Abstract 41

Background: Prenatal polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls 42

(PCBs) exposures may influence children’s neurodevelopment. 43

Objective: To examine the association of prenatal PBDE and PCB exposures with children’s 44 reading skills at ages 5 and 8 years, Full-Scale Intelligence Quotient (FSIQ) and Externalizing 45

Problems at age 8 years. 46

Methods: From 239 mother-child pairs recruited (2003-2006) in Cincinnati, OH, we measured 47 maternal serum PBDE and PCB concentrations, assessed child’s reading skills using the 48

Woodcock-Johnson Tests of Achievement III (WJ-III) at age 5 years and the Wide Range 49

Achievement Test-4 (WRAT-4) at age 8 years, tested FSIQ using the Wechsler Intelligence 50

Scale for Children-IV (WISC-IV), and Externalizing Problems using the Behavioral Assessment 51

System for Children-2 (BASC-2) at age 8 years. We used multiple linear regression to examine 52 the association of prenatal PBDE and PCB concentrations and reading, FSIQ and Externalizing 53

Problems after adjustment for covariates. 54

Results: A 10-fold increase in Sum4PBDEs (BDE-47, -99, -100 and -153) was not significantly 55 associated with reading scores at age 5 years at the p=0.05 level, but was inversely associated 56 with Reading Composite scores (β: -6.2, 95% CI: -11.7, -0.6) and FSIQ (β: -5.3, 95% CI: -10.6, -57

0.02) at age 8 years; it was positively associated with Externalizing Problems score (β: 3.5, 95% 58

CI: -0.1, 7.2) at age 8 years. Prenatal Sum4PCBs (PCB-118, -153, -138-158, and -180) was not 59 significantly associated with child’s reading skills, FSIQ and Externalizing Problems. 60

Conclusion: Prenatal PBDE concentration was inversely associated with reading skills and FSIQ, 61 positively associated with Externalizing Problems at age 8 years. No significant associations 62 were found in prenatal PCB concentration. 63

Keywords: Prenatal exposure; Polybrominated diphenyl ethers (PBDEs); Polychlorinated 65 biphenyls (PCBs); Reading skills; Intelligence; Externalizing problems66

Introduction 67

PBDEs were widely used as flame retardants in the manufacture of electronics, 68 furniture, carpets and textiles, and are detectable in indoor dust, fish, birds, human 69 serum and adipose tissue. The phase-out of PBDEs using in US consumer products 70 began in 2004 (Penta- and Octa-PBDE) and 2013 (Deca-PBDE), PBDEs still exist in 71 the environment and biological samples. PCBs, a group of structurally related organic 72 compounds with inertness and thermal stability, were extensively used in various 73 industrial products before 1979. Although PCBs’ manufacture was banned in 1979 74 due to possible adverse effects in humans, PCBs still persist in the environment and 75 accumulate through the food chain, and seafood consumption is currently the main 76 exposure route in human (Thompson and Boekelheide 2013; Xue et al. 2014). 77

Several previous studies have focused on the association of prenatal PBDE and 78

PCB exposures with child’s overall cognition (Chen et al. 2014b; Stewart et al. 2008; 79

Zhao et al. 2013), and our publication reported that prenatal exposure to PBDEs was 80 associated with cognitive deficits and hyperactivity behaviors in children at age 5 81 years (Chen et al. 2014a). However, few studies have assessed the influence of those 82 chemicals on specific cognitive domains, in particular, the language development and 83 reading skills (Dzwilewski and Schantz 2015). The prenatal period is the most 84 sensitive time window to the influence of environmental factors on cognitive 85 development, when brain development involves neuronal proliferation, differentiation 86 and migration (Kang et al. 2011; Stiles and Jernigan 2010). Reading ability is central 87 to educational attainment and academic achievement, and is predominantly influenced 88 by the development in the brain reading region (Houston et al. 2014). Studies have 89 reported that child’s reading ability was impaired by prenatal maternal smoking 90 exposure (Cho et al. 2013). Externalizing problems are symptoms or signs of 91 neurodevelopment, including hyperactivity, oppositional and aggressive behavior, as 92 well as conduct problems. The purpose of this study was to test the hypothesis that 93 prenatal exposures to PBDEs and PCBs are associated with the poorer reading 94 abilities, lower intelligence and more externalizing problems in children. 95

Methods 97

Study subjects. The Health Outcomes and Measures of the Environment (HOME) 98

Study is a prospective birth cohort recruited between March 2003 and February 2006 99 in Cincinnati, OH, as previously described (Geraghty et al. 2008). The study enrolled 100

468 pregnant women at 16±3 weeks gestation, who were 18 years of age or older, 101 living in Cincinnati, OH, not diagnosed with diabetes, hypertension or reported 102 infection with Human Immunodeficiency Virus, and did not take medicine for 103 seizures or thyroid disorders during pregnancy. Of the 468 women, 389 remained in 104 the study and delivered live-born singletons. For the current study, we excluded 105 infants with congenital malformations or genetic abnormalities (n=2), mothers with 106 missing chemical measures in serum (n=15), and children lost to follow-up and those 107 did not complete assessment at age 5 or 8 years (n=131). This led to a final sample of 108

239 mother-child pairs for the current analysis. The HOME Study was approved by 109 the Institutional Review Boards at the Cincinnati Children’s Hospital Medical Center 110

(CCHMC) and the Centers for Disease Control and Prevention (CDC). All 111 participants provided their written informed consents. 112

Maternal serum chemicals measurement. Maternal blood samples were obtained at 114

16±3 weeks of gestation, and sera were immediately isolated and stored at -80 °C 115 until shipment on dry ice to the National Center for Environmental Health (NCEH) at 116

CDC for analysis. Maternal sera were analyzed for 10 PBDE congeners (BDE-17, -28, 117

-47, -66, -85, -99, -100, -153, -154, -183) and 36 PCB congeners (PCB-18, -28, -44, -118

49, -52, -66, -74, -87, -99, -101, -105, -110, -118, -128, -138-158, -146, -149, -151, -119

153, -156, -157, -167, -170, -172, -177, -178, -180,-183, -187, -189, -199, -196-203, -120

194, -195, -206, -209) using gas chromatography/isotope dilution high-resolution 121 mass spectrometry (Sjodin et al. 2004). All analytic runs included blank and positive 122 quality control samples. PBDEs and PCBs are expressed as nanogram per gram lipid 123

(ng/g lipid) because they are lipophilic compounds. Serum lipid concentrations were 124 calculated using the Phillips formula summing total cholesterol and triglycerides 125

(Phillips et al. 1989). Values below the limit of detection (LOD) were replaced with 126 the LOD divided by the square root of 2 (Hornung-RW and Reed-LD 1990). 127

Reading ability assessments. We assessed children’s reading skills at age 5 years 129 using the Woodcock-Johnson Tests of Achievement-III (WJ-III) (Woodcock et al. 130

2001). For these analyses, we focused on Basic Reading and Brief Reading scores. 131

Basic Reading is a composite of Letter-Word Identification and Word Attack, and 132 measures sight vocabulary, phonics, and structural analysis. Brief Reading is a 133 composite of Letter-Word Identification and Passage Comprehension, and measures 134 reading concepts and readiness. Letter-Word Identification measures the ability to 135 identify letters and read individual words. Word Attack measures skills in applying 136 phonic and structural analysis to the reading of unfamiliar words. Passage 137

Comprehension tests the ability to match a symbol with an actual picture of the object, 138 to point to the picture represented by a phrase, and to identify a missing key word 139 within a passage based on contextual cues. 140

At age 8 years, we measured each child’s basic academic skills to read words 141 and comprehend sentences using the Wide Range Achievement Test 4 (WRAT-4) 142

(Sayegh et al. 2014). Reading Composite score was assessed in the analysis, which 143 was calculated from the Word Reading and Sentence Comprehension subtests. Word 144

Reading measures letter and word reading, and Sentence Comprehension measures 145 the skills to obtain meaning from words and comprehend ideas and information in 146 sentences. Both WJ-III and WRAT-4 items have a population mean of 100 and a 147 standard deviation of 15. 148

Neurodevelopmental and behavioral assessments. At age 8 years, we administered 150 the Wechsler Intelligence Scale for Children-IV (WISC-IV) to obtain Full scale IQ 151

(FSIQ) (Wechsler 2003), and the Behavioral Assessment System for Children-2 152

(BASC-2) to obtain Externalizing Problems score (Reynolds and Kamphaus 2004). 153

The FSIQ measures cognitive function and has a population mean of 100 and standard 154 deviation of 15. Externalizing Problems offer an assessment of a child’s adaptive and 155 problem behaviors, which includes subscales of aggression and hyperactivity. The 156

Externalizing Problems score has a population mean of 50 and a standard deviation of 157

10, for which higher score suggests non-optimal behavior. All clinical assessments 158 were performed by HOME study staffs who were trained and certified by a 159 developmental psychologist (KY). The assessors conducted the neurobehavioral 160 assessments without knowledge of maternal serum PBDE and PCB concentrations. 161

Statistical analysis. We analyzed the associations between maternal serum PBDE or 163

PCB concentrations and the outcome (reading ability, FSIQ or Externalizing 164

Problems) scores using multiple linear regression. We separately used the following 165 exposure variables in the analyses: 1) the sum of BDE-47, -99, -100, and -153 166

(Sum4PBDEs), the four congeners detected in 91% of the HOME study samples, 2) 167 the sum of PCB-118, -153, -138/158, and -180 (Sum4PCBs), these congeners were 168 detected in 94% of the HOME study samples, and Sum4PCBs was frequently used in 169 other US cohort studies (Alm et al. 2008; Boucher et al. 2009). Considering their 170 right-skewed distributions and over 100-fold exposure ranges, log10 transformed 171

Sum4PBDE and Sum4PCB concentrations were used in this analysis. Covariates for 172 adjustment were identified based on their significant relationship with chemical 173 concentrations and outcome scores using analysis of variance (ANOVA) or t-test for 174 continuous variables, and chi-square test for categorical variables. In the final models, 175 we adjusted for maternal age, race, education, household-income, parity, marital 176 status, smoking status, maternal fish consumption (times of fish-containing meals 177 from baseline visit to child birth), maternal depression, maternal intelligence quotient 178

(IQ), child sex, and the Home Observation for Measurement of the Environment 179

(HOME) score (Totsika and Sylva 2004). Maternal IQ was assessed using the 180

Wechsler Abbreviates Scale of Intelligence, maternal depression was measured using 181 the Beck Depression Inventory-II (Beck et al. 1996) during pregnancy. When the 182 child was 1 year old, study staff visited them and completed the HOME Inventory 183 describing the nurturing environment in the home (Caldwell B and Bradley R 1984). 184

We used the trend test to test the each outcome’s trend by using the median value 185 of each quartile as the independent variable. We also used the Least Absolute 186

Shrinkage and Selection Operator (LASSO) test to simultaneously perform model 187 selection and parameter estimation of each PBDE and PCB congener’s relative 188 contribution to the outcome scores (Tibshirani 1996). To ensure the consistency of 189 results, the analysis was repeatedly run with all available data and with data available 190 at both ages of 5 and 8 years. The statistical analysis was completed with SAS 9.4 191

(SAS Institute Inc., NC, USA), the LASSO test were performed in R 2.11 (R 192

Developmental Team) glmnet packages. We report the regression coefficients and 95% 193 confidential intervals (CI) from the multiple regression analyses. The statistical 194 significance was set at a two-sided p value of 0.05. 195

Results 197

This analysis was based on 239 mothers who were enrolled in the HOME study 198 and provided biological samples for evaluating exposures, and their children who had 199 outcomes of reading ability, FSIQ and Externalizing Problems at age 5 or 8 years. 200

There were no significant difference in either maternal PBDE or PCB concentration 201 measured during pregnancy or demographic characteristics between the 239 subjects 202 included in this analysis and those not included (data not shown). 203

In the HOME study, maternal median Sum4PBDEs concentrations (35.65 ng/g 204 lipid) were slightly lower, the median Sum4PCBs (31.30 ng/g lipid) was a little higher 205 as compared to the median concentrations of the U.S. general pregnant women 206

(NHANES, 2003-2004) (Sum4PBDEs: 43.2 ng/g lipid, Sum4PCBs: 26.8 ng/g lipid) 207

(Woodruff et al. 2011) (Figure 1). Mothers were mainly 25-34 years of age (61%), 208 non-Hispanic White (62%) and married or living with a partner (76%), with college 209 education or above (74%), middle or high level of household income (>$40,000/y, 210

76%); most of them did not drink alcohol (56%) and did not smoke tobacco (85%) 211 during pregnancy. Babies were born at a mean gestational age of 39 ± 5.4 weeks. 212

Mothers who were younger than 25 years of age at enrollment, non-white, with a high 213 school or less education, household-income less than $40,000/y, not married or living 214 alone, were smokers or had higher depression symptom scores (>19), had higher 215 prenatal PBDE and lower PCB concentrations. Additionally, prenatal concentrations 216 of Sum4PBDE and Sum4PCB were comparable for boys and girls (Table 1). 217

Children’s scores of reading, FSIQ and Externalizing Problems were normally 218 distributed. Reading subset items were highly correlated with each other (r=0.58 for 219 composite measures, p<0.001). Reading Composite score was positively correlated 220 with FSIQ (r=0.75, p<0.0001), and not correlated with Externalizing Problems score 221

(r=-0.05, p=0.43) at age 8 years. Reading Composite score and FSIQ were positively 222 associated with maternal age, education, household-income, and inversely associated 223 with maternal depression and HOME scores (Supplement material, Figure S1). 224

Externalizing Problems score was negatively associated with maternal education and 225 household-income, and positively associated with maternal depression and HOME. 226

After adjusting for covariates, each 10-fold increase in Sum4PBDEs 227 concentration was significantly associated with a 6.6-point decrement in Sentence 228

Comprehension score (p=0.038), a 6.2-point decrement in Reading Composite score 229

(p=0.029), and a 5.3-point decrease in FSIQ score (p=0.049). It was marginally 230 associated with a 3.5-point increase in Externalizing Problems score (p=0.057) at 8 231 years of age (Table 2). Sum4PCBs concentration as a continuous variable was 232 positively associated with children’s reading scores, FSIQ and Externalizing Problems 233 at age 8 years, but none of them was statistically significant (Table 2). The Sum4PCBs 234 concentration was correlated with maternal fish consumption during pregnancy 235

(r=0.38, p<0.0001) whereas the Sum4PBDEs concentration was not (r=0.05, p=0.38). 236

Non-significant interaction was found between the fish consumption and serum 237

Sum4PCBs on child’s reading abilities, FSIQ and Externalizing Problems in the 238

HOME study (Data not shown). 239

Trend tests based on quartiles indicated that as the median Sum4PBDEs 240 increased in the quartiles, Reading Composite score was significantly decreased 241

(p=0.04), Externalizing Problems score was statistically increased (p=0.03). No 242 significant trend was found in Brief Reading score and FSIQ with Sum4PBDEs; 243

Neither trend nor association was significant between Sum4PCBs quartiles and 244 reading scores at ages 5 and 8 years, FSIQ and Externalizing Problems scores at age 8 245 years (Figure 2). 246

The specific analysis for each PBDE or PCB congener demonstrated that a 10-247 fold increase of BDE-99 was marginally associated with a 5.1-point decrease (95% CI: 248

-10.3, 0.2) in FSIQ at age 8 years (p=0.058). A 10-fold increase of BDE-100 was 249 significantly associated with a 6.0-point decrease (95% CI: -11.8, -0.3) in Sentence 250

Comprehension score (p=0.039), and a 5.7-point decrease (95% CI: -10.8, -0.6) in 251

Reading Composite score (p=0.028) at age 8 years. A 10-fold increase of BDE-153 252 was significantly associated with a 6.3-point decrease (95% CI: -11.6, -0.9) in 253

Sentence Comprehension score (p=0.022), a 5.4-point decrease (95% CI:-10.1, -0.7) 254 in Reading Composite score (p=0.026), and a 3.9-point increase (95% CI: 0.8, 6.9) in 255

Externalizing Problems score (p=0.014) at age 8 years. No significant associations 256 were found between PCB congeners and children’s reading abilities, FSIQ and 257

Externalizing Problems (Table 3). The results were similar when we ran the analysis 258 with all available data and data available at both 5 and 8 years of age. The findings of 259 the LASSO analysis were consistent with the results of the multiple linear regression, 260 and are seen in Table S4. 261

Discussion 263

In this study, we comprehensively assessed child’s various aspects of 264 neurodevelopment, including reading abilities, intelligence and behavior, and 265 highlighted that the reading ability was an important aspect of neurodevelopment, 266 which was influenced by prenatal chemical exposure. After adjustment, prenatal 267

PBDE concentration was inversely associated with reading skills and FSIQ, and 268 positively associated with externalizing problems in children at age 8 years. Prenatal 269

PBDE concentration was not associated with reading skills at age 5 years; Prenatal 270

PCB concentration was not statistically associated with reading abilities, FSIQ and 271 externalizing problems in children at age 5 or 8 years. Prenatal PBDE and PCB 272 concentrations were measured in maternal serum samples at approximately 16 weeks 273 of gestation, when blood volume expansion and hyperlipidemia status have not been 274 established (Bloom et al. 2007; Faupel-Badger et al. 2007), thus the impact of 275 physiological variation during pregnancy on prenatal serum PBDE and PCB 276 concentrations is less important in this study. 277

No evidence is reported on the association of prenatal PBDE and PCB 278 concentrations with child’s reading ability so far. This study highlights reading ability 279 as a key outcome, and found that prenatal PBDE concentration was inversely 280 associated with child’s reading ability at age 8 years after adjustment for covariates, 281 but was not associated with reading ability at age 5 years. Reading ability is an aspect 282 of neurodevelopment, which was significantly influenced by the neurodevelopment 283 maturity, such as brain area and volume (Spann et al. 2014), the ratio of neuron to 284 glial cells, cell integrity and inter-neuronal communication underlying the frontal and 285 temporal cortices (O'Muircheartaigh et al. 2014). The results in the HOME study 286 showing the association of prenatal PBDE with decrements in FSIQ and increment in 287 externalizing problems were remarkably consistent with previous studies. In the 288

HOME study, the decrement in FSIQ (-5.3 [95% CI: -10.6, -0.1]) and increment in 289 externalizing problems (3.5 points [95% CI: -0.1, 7.2]) at age 8 years with a 10-fold 290 increase in maternal serum Sum4PBDEs concentration are consistent with our 291 previous report for FSIQ (β: -4.5, 95% CI: -8.8, -0.1) and externalizing behavior 292 problems (β: 2.6, 95% CI: -0.4, 5.5) at age 5 years with maternal serum BDE-47 293 concentration (Chen et al. 2014a), and are also consistent with other two reports 294

[California farming community cohort: FSIQ (β: -4.7, 95% CI: -9.4, 0.1) and 295

Attention Deficit/Hyperactivity Disorder (ADHD) index (β: 2.9, 95% CI: -0.7, 5.2) at 296 age 7 years with maternal Sum4PBDEs; World Trade Center cohort: FSIQ (β: -5.5, 95% 297

CI: -10.8, -0.2) at age 4 years related to cord BDE-47 (derived from original estimate 298 of natural log BDE-47)] (Eskenazi et al. 2013; Herbstman et al. 2010). Maternal 299 serum PBDE concentration was associated with more externalizing problems (BDE-300

100, β: 0.31), worse fine manipulative abilities (BDE-154, β: -0.30) in 62 healthy 301

Dutch children aged 5-6 years (Roze et al. 2009). 302

Several mechanisms underlying PBDE-mediated developmental neurotoxicity 303 have been suggested in rats and mice neonatal exposure to PBDE (Johansson et al. 304

2008; Viberg et al. 2003, 2005, 2007; Zhang et al. 2013). BDE-47 and BDE-153 305 changed hippocampus morphology and ultra structure in rats (He et al. 2009; Zhang et 306

al. 2013). BDE-47 and BDE-99 damaged rats or mice’s neuron cytoskeletal formation 307 and neuronal maturation by affecting Ca2+/calmodulin-dependent protein kinase II, 308 synaptophysin and cytoskeletal protein expression, decreasing expressions of brain-309 derived neurotrophic factor and anti-apoptotic bcl-2 mRNA and protein (Alm et al. 310

2008; Blanco et al. 2011; Viberg 2009). PBDEs induced oxidative stress, DNA 311 damage and apoptosis (He et al. 2008; Tagliaferri et al. 2010), disrupted intercellular 312

Ca2+ homeostasis (Gassmann et al. 2014), affected neuron cell signal transduction 313

(Fan et al. 2010) and voltage-gated sodium channels ex vivo (Xing et al. 2010). 314

Moreover, PBDEs suppressed thyroid receptor-mediated transcription (Ibhazehiebo et 315

al. 2011), impair rats’ synaptic plasticity (Xing et al. 2009), inhibited human neural 316 progenitor cells migration and differentiation into neurons and oligodendrocytes 317

(Schreiber et al. 2010). All of the above provided potential mechanisms underlying 318

PBDE-mediated developmental neurotoxicity. 319

PCBs are among the most well-studied endocrine-disrupting chemicals, and can 320 cause rat pups’ hyperactivity (Lesmana et al. 2014), induce oxidative stress and 321 increase apoptosis in the developing rat brain (Yang and Lein 2010). In the HOME 322 study, prenatal Sum4PCBs concentration (median: 31.30 ng/g lipid) was not 323 associated with reading skills, FSIQ and externalizing problems in children at age 8 324 years after adjusting for maternal fish consumption and other covariates. This findings 325 was inconsistent with the Oswego cohort study showing an inverse association 326 between placenta Sum4PCBs concentration (1.5 ng/g wet) and FSIQ (β=-0.167, 327 p=0.021) and Verbal IQ (β=-0.213, p=0.003) in 9-year-old children (Stewart et al. 328

2008). The LASSO test revealed positive associations of Sum4PCBs or some PCB 329 congeners with child’s neurodevelopment as well in this study. Consumption of 330 contaminated fish and other food is the main source of human exposure to PCBs 331

(Braun et al. 2014). Fish contains beneficial nutrients for fetal development and 332 maternal health, including polyunsaturated fatty acids (PUFAs) and others, but also 333 may contain harmful contaminants such as PCBs and methylmercury (MeHg). In the 334

HOME study, maternal serum PCB concentrations were positively correlated with 335 fish consumption, yet non-significant interaction was found between maternal serum 336

Sum4PCBs and fish consumption on outcomes. 337

This study has notable strengths and limitations. We comprehensively focused 338 on children’s reading abilities, an often overlooked facet of neurodevelopment, and 339

FSIQ and externalizing problems, in a prospective birth cohort, and assessed chemical 340 exposure during the sensitive prenatal period using maternal serum chemical 341 concentrations, and successfully followed up children into school age when 342 neurodevelopment measures stabilize. We tested the trend of outcome with maternal 343 serum PBDE and PCB concentrations in quartile analysis. One limitation is that we 344 did not have the measurements of serum PUFAs, the omega-3 docosahexaenoic acid, 345 and MeHg in this analysis. These may be correlated with co-exposure to PCBs 346 through fish consumption and neurodevelopmental outcomes and outcomes and thus 347 may confound our analysis. Another limitation is that possible random finding in 348 multiple comparisons without corrections, which cannot be entirely ruled here. While 349 there are statistical methods such as Bonferroni correction to control for multiple 350 comparisons, they tend to be stringent and neglect the correlation between 351 neurodevelopmental outcomes. Finally, we had limited sample size at ages 5 and 8 352 years, thus limiting our statistical power and precision. 353

In conclusion, prenatal PBDE concentration was inversely associated with 354 children’s reading abilities and FSIQ at age 8 years, positively associated with 355 externalizing problems at age 8 years. Prenatal PCB concentration was not 356 significantly associated with child’s reading abilities, FSIQ and externalizing behavior 357 problem. These findings highlight the importance of understanding the potential 358 impact that being exposed to persistent organic chemicals during pregnancy can have 359 on children’s neurodevelopment during early childhood, alert people to protect child’ 360 neurodevelopment from environmental exposure during the entire gestation by 361 minimizing exposure to persistent organic chemicals. Additionally, reading ability is 362 an aspect of neurodevelopment and should be assessed in further studies. 363

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Tables: 538

Table 1 Participant’s demographic characteristics, maternal serum PBDE and PCB 539 concentrations, and child’s scores of reading, FSIQ and Externalizing Problems [mean ± SD or n 540 (%)] 541

Maternal and Child’s Characteristics N (%) Log10Sum4PBDEs Log10Sum4PCBs Maternal age (yrs) 28.38 ± 5.75

<25 yrs 57 (23.55) 1.64±0.31 1.28±0.24 25-34 yrs 150 (61.98) 1.59±0.46 1.53±0.24

>=35 yrs 35 (14.46) 1.40±0.34 1.75±0.21 Maternal education High school or less 61 (25.21) 1.72±0.38 1.38±0.32 Some college or 2 year degree 62 (25.62) 1.58±0.33 1.47±0.20 Bachelor’s 74 (30.58) 1.50±0.44 1.56±0.25 Graduate or professional 45 (18.60) 1.51±0.52 1.63±0.27 Maternal race Non-Hispanic White 150 (61.98) 1.52±0.42 1.55±0.24 Non-Hispanic Black and Others 92 (38.02) 1.68±0.41 1.43±0.32 Maternal parity Nulliparous 111 (45.87) 1.53±0.43 1.54±0.31 Parity=1 71 (29.34) 1.56±0.38 1.49±0.24 Parity>1 60 (24.79) 1.66±0.45 1.49±0.23 Marital status Married or living with partner 184 (76.03) 1.53±0.43 1.55±0.25 Not married, living alone 58 (23.97) 1.72±0.36 1.38±0.31 Household income

<$40,000/y 58 (23.97) 1.73±0.35 1.35±0.29 $40,000-79,999/y 121 (50.00) 1.57±0.44 1.55±0.25

>=$80,000/y 63 (26.03) 1.42±0.39 1.58±0.26 Maternal alcohol drinking Never drinking 136 (56.20) 1.56±0.43 1.48±0.26

<1/month drinking 75 (30.99) 1.59±0.41 1.54±0.24 >1/month or binge 31 (12.81) 1.59±0.43 1.60±0.37

Maternal smoking Non smoking (serum cotinine < 1 ng/ml) 206 (85.12) 1.55±0.42 1.52±0.27

Secondhand tobacco smoking (1 ng/ml=<serum cotinine <

10ng/ml) 19 (7.85) 1.63±0.32 1.32±0.21

Active smoking (serum cotinine >=10 ng/ml) 17 (7.02) 1.91±0.43 1.58±0.28

HOME inventory score at 1 year home visit 39.14 ± 5.44

>=40 146 (64.60) 1.50±0.43 1.57±0.26 35-39 41 (18.14) 1.74±0.39 1.46±0.22

<35 39 (17.26) 1.69±0.40 1.33±0.28

Maternal Full Scale IQ 105.98 ± 15.28 Maternal depressive symptoms at 1 year home visit 9.92 ± 6.67

Minimal or mild 221 (91.70) 1.55±0.42 1.53±0.27 Moderate or severe 20 (8.30) 1.81±0.40 1.37±0.23 Child sex

Male 109 (45.04) 1.53±0.42 1.52±0.26 Female 133 (54.96) 1.61±0.42 1.50±0.28 Reading Scores WJ-III Score at age 5 years (n=203)

Basic Reading (a+c) 109.94 ± 17.64

Brief Reading (a+b) 104.44 ± 16.83

a. Letter Word 106.12 ± 15.57

b. Passage Comprehension 100.66 ± 11.07

c. Word Attack 118.97 ± 13.28

WRAT-4 Score at age 8 years (n=232)

Reading Composite (d+e) 108.27 ± 15.40

d. Word Reading 107.63 ± 14.24

e. Sentence Comprehension 107.63 ± 17.88

FSIQ at age 8 years (n=231) 102.10 ± 15.71 Externalizing Problems at age 8 years (n=239) 49.54 ± 9.40

Table 2 Adjusted β coefficients (95% CIs) in the multiple linear regressions of child’s reading scores, FSIQ and Externalizing Problems score with prenatal serum 547

Sum4PBDEs and Sum4PCBs concentrations (ng/g lipid) 548

Chemicals WJ-III Score (n=203) at age 5 years WRAT-4 Score (n=232) at age 8 years WISC-IV (n=231) at age 8 years

BASC-2 (n=239) at age 8 years

Basic Reading Brief Reading Reading Composite FSIQ Externalizing Problems

Log10Sum4PBDEs -3.1 (-10.1, 3.9)

-2.8 (-9.9, 4.3) -6.2

(-11.7, -0.6)* -5.3 (-10.6, -0.1)*

3.5 (-0.1, 7.2)#

Log10Sum4PCBs 6.7 (-4.8, 18.3)

6.5 (-5.2, 18.2) 7.0

(-2.2, 16.2) 1.1 (-7.9, 10.0)

0.5 (-5.7, 6.7)

^: Adjusted for maternal age, education, race, IQ, household-income, parity, marital status, maternal smoking (serum cotinine concentrations), depression, fish consumption, 549 child sex, and HOME score. CIs: confidential intervals; “*”: p< 0.05, “#”: p< 0.10. 550

Table 3 Adjusted β coefficients (95% CIs) in the multiple linear regressions of child’s reading scores, FSIQ and Externalizing Problems score with prenatal serum 560 concentrations of PBDE and PCB congeners (ng/g lipid) 561

Chemicals

WJ-III score (n=203) at age 5 years

WRAT-4

score (n=232) at age 8 years

WISC-IV

score (n=231) at age 8 years

BASC-2 score (n=239) at age 8 years

Basic Reading

Brief Reading

Reading Composite

FSIQ Externalizing Problems

Log10

BDE-47

-1.2 (-8.0, 5.6)

-0.9 (-7.7, 6.0)

-4.8 (-10.1, 0.6)

3.7 (-8.8, 1.5)

2.1 (-1.4, 5.6)

Log10

BDE-99

-3.8 (-10.6, 3.0)

-3.2 (-10.0, 3.7)

-4.2 (-9.7, 1.3)

-5.1 (-10.3, 0.2)#

2.8 (-0.8, 6.4)

Log10

BDE-100

-3.1 (-9.7, 3.4)

-3.1 (-9.7, 3.5)

-5.7 (-10.8, -0.6)*

-4.6 (-9.5, 0.2)#

2.6 (-0.7, 6.0)

Log10

BDE-153

-4.8 (-10.5, 1.0)

-4.7 (-10.6, 1.1)

-5.4 (-10.1, -0.7)*

-3.6 (-8.2, 0.9)

3.9 (0.8, 6.9)*

Log10 PCB-

2.0 (-7.8, 11.7)

1.2 (-8.7, 11.1)

3.8 (-4.2, 11.8)

-0.5 (-8.2, 7.2)

-1.2 (-6.6, 4.1)

Log10 PCB-

6.3 (-4.8, 17.4)

7.1 (-4.1, 18.4)

6.3 (-2.5, 15.1)

-0.1 (-8.7, 8.5)

0.8 (-5.1, 6.7)

Log10PCB- 138-158

6.2 (-4.5, 16.8)

5.5 (-5.3, 16.3)

3.5 (-5.1, 12.0)

0.6 (-7.7, 8.9)

0.9 (-4.8, 6.7)

Log10 PCB-

4.4 (-6.3, 15.2)

6.0 (-4.8, 16.8)

6.8 (-1.6, 15.2)

0.7 (-7.5, 8.9)

0.8 (-4.9, 9.4)

^: Adjusted for maternal age, education, race, IQ, household-income, parity, marital status, maternal smoking (serum cotinine concentrations), depression, fish consumption, 562 child sex, and HOME score. CI: confidential intervals; “*”:p< 0.05, “#”:p< 0.10. 563

Figure legends: 565

Figure 1. Concentrations of PBDE and PCB congeners in the pregnant women of HOME Study and NHANES (2003-2004) (Median and interquartile range). 567

^: Bars present the interquartile range (IQR) of chemical concentrations in the HOME Study. Medians of chemical concentrations in the NHANES pregnant women (3004-568

2004) were referenced, which did not provide the IQR or p25 and p75 percentiles (Woodruff et al. 2011). 569

National Health and Nutrition Examination Survey; BDE-47, -99, 100 and -153 are the congeners of polybrominated diphenyl ethers (PBDEs); PCB-118, -153, -138-158, -570

180 are the congeners of polychlorinated biphenyls (PCBs). 571

Limit of detection (LOD) for NHANES pregnant women (2003-2004) is 4.2, 5.0, 1.4 and 2.2 ng/g lipid for BDE-47, -99, 100, and -153; 0.6 and 1.1 ng/g lipid for PCB-118, -572

153, 0.4 ng/g lipid for PCB-180, PCB-138-158. 573

Figure 2 Trend and association of child’s reading scores, FSIQ and Externalizing Problems scores with prenatal Sum4PBDEs and Sum4PCBs concentrations quartiles 576

^: Adjusted for maternal age, education, race, IQ, home income, parity, married status, smoking (maternal serum cotinine), fish consumption, depression, and child sex and 577

HOME score. a: The trend and association of child’s reading scores, FSIQ and Externalizing Problems scores with prenatal Sum4PBDEs; b: The trend and association of 578 child’s reading scores, FSIQ and Externalizing Problems scores with prenatal Sum4PCBs. The quartile cutoffs were < 20.70, 20.70-35.64, 35.65-76.00, and ≥ 76.00 ng/g lipid 579 for Sum4PBDEs, and <21.50, 21.50-31.29, 31.30-42.80, and ≥ 42.80 ng/g for Sum4PCBs, respectively. The score in the 1st quartile is the reference. “*”: p< 0.05, “#”: p< 0.10. 580

Supplemental materials: 582

Table S1 Coefficients in LASSO test of maternal chemical concentrations to children’s reading scores, FSIQ and Externalizing Problems score at age 8 years 583

Models Chemicals Reading Composite FSIQ Externalizing Problems

Model 1 Log10Sum4PBDEs -5.6 -3.8 2.1 Log10Sum4PCBs 6.5 0.5 -

Model 2

Log10BDE-47 - - -

Log10BDE-99 - -4.1 0.5 Log10BDE-100 -3.8 - - Log10BDE-153 -1.6 -0.5 2.2 Log10PCB-118 1.0 - -0.3 Log10PCB-138-158 - - - Log10PCB-153 6.2 - - Log10PCB-180 0.5 1.3 -

^: Adjusted for maternal age, education, race, IQ, household-income, parity, marital status, maternal smoking (serum cotinine concentrations), depression, fish consumption, 584 child sex, and HOME score. It can not yield 95% Cis when selecting variables and estimating parameters. 585

Model 1: LASSO regression with Log10Sum4PBDEs and Log10Sum4PCBs after adjustment. 586

Model 2: LASSO regression with Log10 transformed PBDE congeners (BDE-47, -99, -100, and -153) and PCB congeners (PCB-118, -138-158, -153, and -180) after 587 adjustment. 588

Figures: 591

Figure S1 Children’s Reading Composite scores at age 8 years by maternal participants and children’s demographic characteristics in the HOME Study (“*”: p < 0.05 593 comparing to the first subgroup given in this graph.) 594

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